Composition for treating diabetes or obesity

Abstract

The present invention relates to a composition for treating diabetes or obesity. In one embodiment, the present invention relates to peptide analogs of pancreatic polypeptide (PP) for use in the treatment of diabetes or obesity. Also disclosed are methods of treating diabetes or obesity, as well as the use of the peptide analogs according to the invention for the preparation of a medicament for the treatment of diabetes or obesity.

Description

Field of the Invention

[0001] The present invention relates to a composition for treating diabetes or obesity. In one embodiment, the present invention relates to a peptide analogue of pancreatic polypeptide (PP) for treating diabetes or obesity. Also disclosed are methods of treating diabetes or obesity, and the use of the peptide analogue according to the present invention in the preparation of a medicament for treating diabetes or obesity. Background of the Invention

[0003] Peptides of the neuropeptide Y (NPY) family are composed of three bioactive peptide hormones, namely NPY, peptide YY (PYY), and pancreatic polypeptide (PP). Each of these peptides consists of 36 amino acids with significant sequence homology, and their biological actions are mediated through binding and interaction with neuropeptide Y receptors (NPYRs) (including NPY1 receptor, NPY2 receptor, NPY4 receptor, and NPY5 receptor). Although NPY is considered to have important physiological roles in the central nervous system (CNS), recent research on NPY peptides has been related to the potential metabolic benefits of PYY peptides.

[0004] The dipeptidyl peptidase-4 (DPP-4) N-terminal degradation products of PYY(1-36) and PYY(3-36) are an NPY2R-selective agonist and have a defined satiety effect in animals and humans. Initial attempts to utilize the therapeutic potential of PYY(3-36) and NPYR2 activation in humans were frustrated by adverse gastrointestinal side effects. However, a sustained-release NPY2R agonist has recently been characterized, which has a retained satiety effect in humans but significantly reduced adverse effects, indicating that translation to the clinic can still be achieved. In addition, the beneficial effects of continuous activation of the NPYR1 receptor by native full-length PYY(1-36) have recently been recognized at the level of the endocrine pancreas. Thus, in preclinical models of diabetes, extended NPY1R activation improved pancreatic β-cell growth, survival, and overall secretory function, as well as aspects of islet cell transdifferentiation events, with benefits shown to translate to isolated human islets. In summary, PYY molecules provide anti-obesity therapeutic potential through modulation of the NPYR2 receptor by PYY(3-36), and anti-diabetic effects through positive regulation of NPYR1 by PYY(1-36). Unfortunately, due to the different receptor activation profiles of PYY(1-36) and PYY(3-36), these metabolic benefits appear to be mutually exclusive for PYY compounds.

[0005] An ideal regimen leveraging the therapeutic benefits of NYPR activation would include both anti-obesity and anti-diabetic benefits in the same compound. In this regard, pancreatic polypeptide (PP), which preferentially activates NPY4R, has a good satiety effect in both mice and humans. In fact, the importance of NPY4R signaling for energy homeostasis was exemplified in mice overexpressing PP, which showed reduced energy intake and weight gain. Additionally, administration of the specific β-cell toxin streptozotocin (STZ) to mice increased local islet PP expression, which may be an adaptive response that helps maintain or restore normal islet architecture. Consistently, in vitro observations revealed that PP plays a protective role against STZ-induced pancreatic β-cell DNA damage. Thus, PP and related NPY4R regulation appear to have the potential to combine the anti-obesity and anti-diabetic benefits of NPYR signaling. To expand on these important observations and advance a possible new treatment regimen for diabetes, we must continue our preliminary research on this compound.

[0006] Diabetes and obesity are reaching epidemic proportions, and despite the current range of available drugs, many people still do not have adequate metabolic control. Thus, there is an urgent need for new and more effective treatment options. Consequently, there is a significant commercial opportunity for new and effective anti-diabetic and anti-obesity agents with novel modes of action. Summary of the Invention

[0008] According to a first aspect of the present invention, there is provided a peptide analogue of pancreatic polypeptide (PP) for the treatment of diabetes or obesity.

[0009] According to a second aspect of the present invention, there is provided a method for the treatment of diabetes or obesity, the method comprising the step of administering to a subject in need thereof a peptide analogue of pancreatic polypeptide (PP).

[0010] Optionally, the peptide analogue of pancreatic polypeptide (PP) has the amino acid sequence APLEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY.

[0011] Optionally, the peptide analogue of pancreatic polypeptide (PP) has the amino acid sequence APLEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY as defined in SEQ ID NO:1.

[0012] Optionally, the peptide analogue of pancreatic polypeptide (PP) comprises SEQ ID NO:1 and at least one amino acid substitution or modification.

[0013] Optionally, the peptide analogue of pancreatic polypeptide (PP) comprises SEQ ID NO:1 and at least one amino acid substitution.

[0014] Optionally, the peptide analogue of pancreatic polypeptide (PP) comprises SEQ ID NO:1 and at least one amino acid substitution, wherein at least one amino acid substitution comprises substitution of proline with leucine.

[0015] Optionally, the peptide analogue of pancreatic polypeptide (PP) comprises SEQ ID NO:1 and at least one amino acid substitution, wherein at least one amino acid substitution comprises substitution of proline with leucine at position 3 of SEQ ID NO:1.

[0016] Optionally, the peptide analogue of pancreatic polypeptide (PP) comprises SEQ ID NO:1 and at least one amino acid substitution, wherein at least one amino acid substitution comprises substitution of proline with leucine at position 3 of SEQ ID NO:1, as defined by SEQ ID NO:2.

[0017] Optionally, the peptide analogue of pancreatic polypeptide (PP) has the amino acid sequence APPEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY.

[0018] Optionally, the peptide analogue of pancreatic polypeptide (PP) has the amino acid sequence APPEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY, as defined by SEQ ID NO:2.

[0019] Preferably, the peptide analogue of pancreatic polypeptide (PP) is SEQ ID NO:2.

[0020] According to a third aspect of the present invention, there is provided a composition comprising the peptide analogue according to the first or second aspect of the present invention and a pharmaceutically acceptable carrier.

[0021] Optionally, the composition comprises a pharmaceutically effective amount of the peptide analogue according to the first or second aspect of the present invention and a pharmaceutically acceptable carrier.

[0022] Optionally, the composition can be administered parenterally.

[0023] Optionally, the composition can be administered parenterally, for example by injection (such as by intramuscular injection or by subcutaneous injection).

[0024] Optionally, the composition can be provided as a liquid composition.

[0025] Optionally, the use includes administering the peptide analogue according to the first or second aspect of the present invention or the composition according to the third aspect of the present invention.

[0026] Optionally, the use includes administering to a subject a peptide analogue according to the first or second aspect of the present invention or a composition according to the third aspect of the present invention.

[0027] Optionally, the use includes administering to a subject suffering from diabetes a peptide analogue according to the first or second aspect of the present invention or a composition according to the third aspect of the present invention.

[0028] Even more optionally, the use includes administering to a subject suffering from obesity a peptide analogue according to the first or second aspect of the present invention or a composition according to the third aspect of the present invention.

[0029] Optionally, the use includes administering a pharmaceutically effective amount of a peptide analogue according to the first or second aspect of the present invention or a composition according to the third aspect of the present invention.

[0030] Optionally, the use includes administering 0.25 nmol / kg body weight - 25.00 nmol / kg body weight of a peptide analogue according to the first or second aspect of the present invention or an equivalent amount of a composition according to the third aspect of the present invention.

[0031] Still more optionally, the use includes administering 2.5 nmol / kg body weight - 25.00 nmol / kg body weight of a peptide analogue according to the first or second aspect of the present invention or an equivalent amount of a composition according to the third aspect of the present invention.

[0032] Still more optionally, the use includes administering 25.00 nmol / kg body weight of a peptide analogue according to the first or second aspect of the present invention or an equivalent amount of a composition according to the third aspect of the present invention.

[0033] According to a fourth aspect of the present invention, there is provided a method for treating diabetes or obesity, the method comprising administering a peptide analogue according to the first or second aspect of the present invention or a composition according to the third aspect of the present invention.

[0034] According to a fifth aspect of the present invention, there is provided the use of a peptide analogue according to the first or second aspect of the present invention in the preparation of a medicament for treating diabetes or obesity.

[0035] Optionally, the diabetes is type 2 diabetes.

[0036] Optionally or additionally, the peptide analogue is used for reducing food intake. Still optionally or additionally, the peptide analogue is used for reducing appetite.

[0037] Optionally, the peptide analogue is used for reducing blood glucose and optionally for increasing insulin levels.

[0038] Optionally, the peptide analog is used to improve glucose homeostasis. Optionally, the peptide analog is used to increase pancreatic insulin content.

[0039] Optionally, the peptide analog is used to increase the total islet area and β-cell area.

[0040] Optionally, the peptide analog is used to reduce the islet α-cell area.

[0041] Optionally, the peptide analog is used to reduce glucagon-positive stained cells.

[0042] Optionally, the peptide analog is used for β-cell dedifferentiation.

[0043] Optionally, the peptide analog is used for transdifferentiation into non-insulin-positive islet cell types. Brief Description of the Drawings

[0045] Figure 1 The figure shows the effects of the PP peptide on cell viability, insulin release, proliferation, and apoptosis of rodent BRIN-BD11 cells. (A) Cells were incubated with the test peptide (10 -8 M - 10 -6 M) for 18 h before adding MTT. (B, C) Cells were incubated with (B) 16.7 mM glucose (20 min) or (C) 16.7 mM glucose supplemented with alanine (10 mM) alone (20 min), or 16.7 mM glucose supplemented with alanine (10 mM) together with the test peptide (10 -12 M - 10 -6 M) (20 min), and insulin secretion was evaluated by RIA. (D, E) To evaluate the effects on BRIN BD11 β-cell proliferation and apoptosis and receptor selectivity, cells were incubated with the test peptide alone (10 -8 M - 10 -6 M), or cells were incubated with the test peptide (10 -8 M - 10 -6 M) together with the cytokine mixture (IL-1β 100 U / ml, IFN-γ 20 U / ml, TNF-α 200 U / ml) in the absence or presence of the Y4 antagonist (S)-VU0637120 (10 -5 M) for 18 h as specified. Values are mean ± SEM (n = 8). Compared to the appropriate control cultures, i.e., (A, D, E) medium alone or (B, C) 16.7 mM glucose, *p < 0.05, **p < 0.01, and ***p < 0.001. Compared to (C) 10 mM alanine or (E) the cytokine mixture, ΔP < 0.05, ΔΔ P < 0.01, ΔΔΔP < 0.001. Compared with the effects in the absence of the NPYR4 antagonist (S)-VU0637120, Φ P < 0.05, ΦΦ P < 0.01, ΦΦΦ P < 0.001.

[0046] Figure 2 The figure shows the effects of the PP peptide on food intake and glucose homeostasis in mice. (A, B) The test peptide (25 nmol / kg bw, i.p.) was administered 0 h (A) or 4 h (B) before assessing food intake in overnight (16 h) fasted mice. (C, D) In mice fasted for 16 h, the test peptide (25 nmol / kg bw, i.p.) was co-administered with glucose (18 mmol / kg bw, i.p.), and the corresponding 0 - 60 min AUC values are also shown. Values are the mean ± SEM of 8 mice. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the appropriate saline (A, B) or glucose alone (C, D) controls.

[0047] Figure 3 The figure shows the effects of twice-daily [P 3 PP treatment for 28 days on (A) percentage change in body weight, (B) energy intake, (C) blood glucose, and plasma (D) insulin, (E) glucagon, and (F) glucose:insulin ratio in HFF-STZ mice. (A - C) Parameters were regularly evaluated during 28-day treatment of HFF-STZ mice with twice-daily [P 3 PP (25 nmol / kg bw, i.p.). (D - F) Plasma insulin and glucagon concentrations and glucose:insulin ratio were measured on day 28. Values are the mean ± SEM of 8 mice. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to HFF-STZ controls.

[0048] Figure 4 The figure shows the effects of twice-daily [P 3 PP treatment for 28 days on (A, B) glucose tolerance, (C, D) insulin secretion, (E, F) insulin sensitivity, and pancreatic (G) insulin and (H) glucagon content in HFF-STZ mice. In HFF-STZ mice, after treatment with [P 3All parameters were evaluated after 28 days of twice-daily treatment with [P]PP (25 nmol / kg bw, i.p.). In overnight-fasted mice, blood glucose (A, B) and plasma insulin (C, D) were measured before and after a single administration of glucose (18 mmol / kg, i.p.) at t = 0 min. Blood glucose was evaluated at t = 0 min after insulin administration (15 U / kg bw, i.p.) in non-fasted mice (E, F). Pancreatic insulin and glucagon contents were measured by RIA or ELISA, respectively (G, H). Values are the mean ± SEM of 8 mice. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the HFF-STZ control.

[0049] Figure 5 The figure shows the effect of twice-daily [P 3 PP treatment on islet morphology in HFF-STZ mice. After 28 days of twice-daily treatment of HFF-STZ mice with [P 3 PP (25 nmol / kg bw, i.p.), (A) islet area, β-cell area, and α-cell area, (B) α:β ratio, (C) islet size distribution, (D) percentage of glucagon-positive centrally stained cells, and β-cell (E) proliferation and (F) apoptosis were evaluated. Islet morphology was evaluated using Cell F Image Analysis software, where β-cell proliferation and apoptosis were measured by Ki-67 or TUNEL staining, respectively. Representative islet images (×40 magnification) show (G) insulin (red) and glucagon (green), (H) insulin (red) and Ki-67 (green), or (I) insulin (red) and TUNEL (green) from each treatment group (G-I). Values are the mean ± SEM of 8 mice. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the HFF-STZ control.

[0050] Figure 6 The figure shows the effect of twice-daily [P 3 PP (25 nmol / kg bw, i.p.) treatment on body weight, energy intake, and circulating glucose in streptozotocin (STZ)-induced diabetic Ins1 Cre / + ; Rosa26-eYFP mice. (A) Body weight, (B) % change in body weight, (C) cumulative calorie consumption, (D) blood glucose, (E) non-fasting blood glucose, and (F) fasting blood glucose. Values are the mean ± SEM of 7 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the STZ diabetic control.

[0051] Figure 7 The figure shows the effect of twice-daily [P3 Effect of [P Cre / + PP (25 nmol / kg bw) on the pancreatic morphology of STZ Ins1

[0052] Figure 8 The figure shows twice-daily [P 3 PP (25 nmol / kg bw) on STZ Ins1 Cre / + ; Effect on pancreatic β-cell characteristics in Rosa26-eYFP mice. (A-D) β-cell dedifferentiation (insulin-ve, GFP+ve cells) and (B) β-to-α transdifferentiation (glucagon+ve, GFP+ve cells) were measured using CellF image analysis software. Representative images (40X) of islets from each group of mice show (C) insulin (red), (D) glucagon (red) immunoreactivity with GFP (green) and DAPI (blue). Values are the mean ± SEM of 7 mice. **P < 0.01 and ***P < 0.001 compared with the STZ control.

[0053] Figure 9 The figure shows twice-daily [P 3 PP pancreatic polypeptide (25 nmol / kg bw) on STZ Ins1 Cre / + ; Effect on pancreatic α-cell turnover in Rosa26-eYFP mice. (A) Quantification of α-cell proliferation rate and (B) α-cell apoptosis rate. Representative images (40X) of islets show (C) glucagon (green), Ki-67 (red), and DAPI (blue) or (D) glucagon (red) and TUNEL staining (green); scale bar 100 μm. Values are the mean ± SEM of 6 mice per group, with approximately 50 islets analyzed per group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the appropriate non-diabetic control. Δp < 0.05, ΔΔP < 0.01 compared with the appropriate STZ control.

[0054] Figure 10 The figure shows twice-daily [P 3 PP pancreatic polypeptide (25 nmol / kg bw) on STZ Ins1 Cre / +; Effects on pancreatic β-cell turnover rate in Rosa26-eYFP mice. Quantification of (A) β-cell proliferation rate and (B) β-cell apoptosis rate. Representative images of islets (40×) showing (C) insulin (green), Ki-67 (red), and DAPI (blue) or (D) insulin (red) and TUNEL staining (green); scale bar 100 μm. Values are mean ± SEM of 6 mice per group, with approximately 50 islets analyzed per group. ***p < 0.001 compared to appropriate non-diabetic controls. ΔΔΔP < 0.001 compared to appropriate STZ controls.

[0055] Figure 11 The figure shows twice-daily [P 3 PP (25 nmol / kg bw, i.p.) treatment on streptozotocin (STZ)-induced diabetic Glu CreERT2 ; Effects on body weight, energy intake, and circulating glucose in Rosa26-eYFP mice. (A) Body weight, (B) % change in body weight, (C) cumulative calorie consumption, (D) blood glucose, (E) non-fasting blood glucose, and (F) fasting blood glucose. Values are mean ± SEM of 7 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to STZ diabetic controls.

[0056] Figure 12 The figure shows twice-daily [P 3 PP (25 nmol / kg bw) on STZ Glu CreERT2 ; Effects on pancreatic morphology in Rosa26-eYFP mice. (A-D) Islet area (A), β-cell area (B), α-cell area (C), and α:β ratio (D) were measured using CellF image analysis software. (E) Representative images of islets (40x) from mice in each group showing insulin (red), glucagon (green), and DAPI (blue) immunoreactivity. Values are mean ± SEM of 7 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to STZ controls.

[0057] Figure 13 The figure shows twice-daily [P 3 PP (25 nmol / kg bw) on STZ Glu CreERT2; Effects on pancreatic α-cell characteristics in Rosa26-eYFP mice. (A-D) α-cell transdifferentiation (insulin+ve, GFP+ve cells) and (C) α-dedifferentiation (glucagon+ve, GFP+ cells) were measured using CellF image analysis software. Representative images of islets (40×) show (B) insulin (red) or (D) glucagon (red) immunoreactivity with GFP (green) and DAPI (blue) from each group of mice. Values are mean ± SEM of 7 mice. **p < 0.01 compared with appropriate non-diabetic controls. Δp < 0.05, ΔΔP < 0.01 compared with appropriate STZ controls.

[0058] Materials & Methods

[0059] Peptide

[0060] The PP peptide was synthesized by Synpeptide (Shanghai, China) with 95% purity and internally confirmed by high performance liquid chromatography (HPLC) and MALDI-TOF as previously performed [Gault et al., 2011]. Briefly, peptide samples were injected into an HPLC system (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and then eluted within 240 min using a gradient program from 0.05 / 99.95 (v / v) TFA / water to 0.05 / 19.95 / 80.00 (v / v / v) TFA / water / acetonitrile using a Kinetex C-18 analytical column (150 × 4.60 mm, Phenomenex, Cheshire, UK). Column effluent was monitored by UV absorbance at 214 nm. For peptide mass detection, α-cyano-4-hydroxycinnamic acid was used as the matrix and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Perspective Biosystems, USA) was used in positive detection mode.

[0061] Enzyme stability

[0062] To establish the in vitro stability of PP and [P 3 PP, the peptide (10 μg) was incubated with purified porcine DPP-4 (5 mU in 50 mmol / l triethanolamine / HCl, pH 7.4; Sigma-Aldrich) for 0 - 8 h and the degradation profile was examined using RP-HPLC and MALDI-ToF using the same system as outlined above.

[0063] In vitro effects on insulin secretion, cell viability, receptor selectivity, and β-cell proliferation and survival

[0064] Preliminary studies explored the effects of native PP and [P 3 PP on glucose-induced insulin secretion, β-cell proliferation, cell viability, and survival in BRIN-BD11 cells. Cells were cultured in RPMI 1640 medium (Gibco Life Technologies Ltd) supplemented with 10% v / v fetal bovine serum (Gibco), 1% v / v antibiotics (0.1 mg / ml streptomycin and 100 U / ml penicillin) in 5% atmospheric CO 2 at 37 °C. For insulin secretion experiments, cells were seeded at a density of 150,000 cells / well in 24-well plates (Falcon Ltd). After overnight attachment, the medium was withdrawn, and the cells were then pre-incubated in 1.1 mM glucose KRB buffer for 40 min. The pre-incubation buffer was removed, and 1 ml of KRB test solution containing 16.7 mM glucose and PP test peptide (10 -12 M - 10 -6 M) was added. In a second series of experiments, the effect of the PP test peptide (10 -8 M - 10 -6 M) on insulin secretion was determined after incubation in the presence of 16.7 mM glucose and 10 mM alanine. For all experiments, after a 20-min incubation period, the supernatant was collected and stored at -20 °C until insulin concentration was determined by a well-characterized dextran-coated charcoal radioimmunoassay [Flatt and Bailey, 1981].

[0065] To evaluate the effect of the PP peptide on β-cell viability, BRIN-BD11 cells (40,000 cells / well) were incubated with the test peptide (10 -6 M and 10 -8 M) for 18 h before performing the MTT assay, as previously described [Green et al., 2016]. Briefly, cells were seeded at a cell density of 10,000 cells / well in 96-well plates in RPMI-1640 medium for 24 h, in the absence and presence of the test peptide (10 2 M - 10 -8 M) in a CO -6 incubator for 24 h. After incubation, the cells were supplemented with 20 μl of MTT solution (5 mg / ml) and incubated at 37 °C for 2 h. Then the medium was removed, and the formazan crystals were dissolved using 100 μl of DMSO with agitation of the plate for 10 min. Absorbance was read on a spectrophotometer at an excitation wavelength of 570 nm and an emission wavelength of 630 nm, respectively.

[0066] To examine the PP peptide (10-8 M and 10 -6 M) on β-cell proliferation and prevention of apoptosis, BRIN-BD11 cells were seeded onto sterilized clear glass coverslips (16 mm diameter) and placed in 12-well plates (Falcon Ltd) at a density of 40,000 cells / well, and cultured for 18 h. Unsupplemented medium, GLP-1 (10 -8 M and 10 -6 M), and a cytokine mixture (IL-1β (100 U / mL), IFNγ (20 U / mL), TNFα (200 U / mL)) were used as controls, as appropriate. The cells were then rinsed with PBS and fixed using 4% paraformaldehyde. After retrieving antigens in citrate buffer at 95 °C for 20 min, blocking was performed using 2% BSA for 45 min. For proliferation studies, the coverslips were incubated at 37 °C with rabbit anti-Ki-67 primary antibody (Abcam, ab15580), and then with Alexa 488 secondary antibody. The coverslips were mounted onto polylysine-coated microscope slides and mounted using a 50:50 glycerol:PBS solution, and stored at 4 °C until analysis was required. To determine the ability of the PP peptide to prevent cytokine-induced apoptosis, BRIN BD11 cells were seeded, washed, and fixed as described above, except that the medium was supplemented with the cytokine mixture. The coverslips were then incubated at 37 °C with the TUNEL reaction mixture (Roche Diagnostics Ltd, UK) for 60 min and mounted onto microscope slides as described above. All slides were observed using a fluorescence microscope (Olympus System Microscope, model BX51; Southend-on-Sea, UK), and photographed using a DP70 camera adapter system. The proliferation / TUNEL positive frequency was determined using the cell counter function on ImageJ software and expressed as a percentage of the total cells analyzed, as previously described in our laboratory. In a separate series, a specific Y4 receptor antagonist (10 -5 M, (S)-VU0637120, Glixx Laboratories Inc) was used to evaluate the receptor selectivity of the PP peptide related to β-cell proliferation and anti-apoptotic effects.

[0067] Acute in vivo experiments

[0068] Studies were conducted using adult male Swiss mice (Envigo Ltd, UK) at 12 weeks of age. Mice were housed in a temperature-controlled unit environment (22 ± 2 °C) under a 12-hour light / dark cycle and had free access to drinking water and a maintenance diet (10% fat, 30% protein, and 60% carbohydrates, Trouw Nutrition, UK). In overnight (16 h) fasted mice (n = 8), the acute effects of the PP test peptide on glucose homeostasis and insulin secretion were evaluated after intraperitoneal (i.p.) injection of either glucose alone (18 mmol / kg bw) or glucose (18 mmol / kg bw) in combination with the test peptide (25 nmol / kg bw). To evaluate the effect of the PP peptide on food intake, mice fasted for 16 h (n = 8) received an i.p. injection of saline vehicle (0.9% [w / v] NaCl) or the test peptide (25 nmol / kg bw), and cumulative food consumption was monitored over a 150-minute period. The sustained effect on feeding was examined by injecting the peptide (25 nmol / kg bw) or saline vehicle 4 h before feeding.

[0069] Chronic in vivo experiments

[0070] For the initial study, adult male NIH Swiss mice (12 weeks old) were maintained on a high-fat diet (45% fat, 20% protein, 35% carbohydrates; 26.15 kJ / g total energy percentage; Dietex International Ltd., Witham, UK) for 3 consecutive weeks. After this time period, they were administered an i.p. injection of streptozotocin once a week for 3 weeks, 4 weeks, and 5 weeks (fasted for 4 h, 50 mg / kg bw, dissolved in sodium citrate buffer, pH 4.5). Starting from week 6, diabetic mice (non-fasting blood glucose > 11.1 mmol / l) were grouped (n = 8) and received an intraperitoneal injection twice daily (08:00 and 20:00) of saline vehicle (0.9% [w / v] NaCl) or [P 3 PP (25 nmol / kg bw) for 28 days. Throughout the experiment, the mice continued on the high-fat diet. For the study of transgenic mice (i.e., Ins1 Cre / + ; Rosa26-eYFP mice and Glu CreERT2 ; Rosa26-eYFP mice), diabetes was induced by a multiple low-dose streptozotocin (STZ) injection protocol (fasted for 4 h, 50 mg / kg bw, i.p., in sodium citrate buffer, pH 4.5) for 5 consecutive days. After biochemical confirmation of diabetes development, the mice received an injection twice daily (09:00 and 17:00 h) of saline vehicle (0.9% (w / v) NaCl) or [P 3PP (25 nmol / kg bw) treatment was continued for 11 days. For all studies, cumulative energy intake and body weight were regularly evaluated, and circulating glucose, insulin, and glucagon were measured at the end of the treatment period, as appropriate. In addition, glucose tolerance (18 mmol / kg bw; i.p.; fasted for 18 h) and insulin sensitivity (15 U / kg bovine insulin; i.p.; non-fasted) tests were performed where appropriate. Terminal analysis included dissection of pancreatic tissue, which was processed for quantification of hormone content or islet morphology after acid-ethanol protein extraction or fixation in 4% PFA, respectively. All animal experiments were approved by the Ulster University Animal Ethics Review Committee and were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.

[0071] Biochemical analysis

[0072] Blood samples were obtained from conscious mice via a cut tip on the tail vein and blood glucose was immediately measured using an Ascencia Contour glucometer (Bayer Healthcare Newbury, UK). Blood was collected in cooled heparin / fluoride-coated microcentrifuge tubes (Sarstedt, Numbrecht, Germany) and centrifuged at 12,000 rpm for 15 min using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) to separate plasma. Insulin and glucagon were then measured by in-house radioimmunoassay [Flatt and Bailey, 1981] or commercially available ELISA (EZGLU-30K, Merck Millipore, Burlington, Massachusetts), respectively.

[0073] Immunohistochemistry

[0074] Islet morphology was examined by immunohistochemical staining for insulin (1:400; ab6995, AbCam) or glucagon (1:1000; ab92517, AbCam). Image analysis was performed using Cell F Image Analysis Software (Olympus Soft Imaging Solutions, GmbH, Münster, Germany). To assess islet morphology, the "closed polygon" available within the Cell F Image Analysis Software was used to quantify the area of insulin and glucagon positive staining and expressed as μm 2Calculated islet area / β-cell area / α-cell area. Co-staining of insulin with Ki-67 (1:400; ab15580, AbCam) or TUNEL (In Situ Cell Death Detection Kit, fluorescein; Roche Diagnostics) was used to study β-cell proliferation and apoptosis. For quantification, the number of insulin-positive cells co-expressing Ki-67 or TUNEL was counted separately using ImageJ software, with >80 islets analyzed for each treatment group. For the transdifferentiation study in transgenic mice, co-staining was performed using insulin or glucagon with GFP as above (1:400; ab5450, AbCam). In all cases, after incubation with the primary antibody, the following secondary antibodies were used as appropriate: Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit (1:400; ThermoFisher Scientific). The slides were finally incubated with DAPI and then mounted using a fluorescence microscope (Olympus system microscope, model BX51) equipped with DAPI (350 nm), FITC (488 nm), and TRITC (594 nm) filters and an Olympus XM10 camera system for imaging.

[0075] Statistical analysis

[0076] Statistical tests were performed using GraphPad PRISM software (version 5.0). Values are expressed as mean ± SEM. Analysis of inter-group comparisons was performed using one-way ANOVA with Bonferroni post-test, two-way ANOVA with Bonferroni post-test, or Student's unpaired t-test, as appropriate. Differences were considered significant if p < 0.05. Examples

[0077] The embodiments of the present invention will now be described by way of non-limiting examples.

[0078] Example 1

[0079] In vitro DPP-4 stability

[0080] As expected, incubation of PP with DPP-4 resulted in the production of the N-terminally cleaved product PP(3-36) (Table 1). In contrast, 3 PP was completely resistant to DPP-4-mediated degradation (Table 1).

[0081] Table 1. Amino acid sequence and DPP-4 stability of 3 PP

[0082]

[0083] Native PP and [P 3 PP sequences using single-letter amino acid symbols. [P 3 Amino acid substitutions in [P

[0084] Example 2

[0085] Effect of PP peptides on cell viability, insulin release, receptor selectivity, and β-cell proliferation and prevention of cytokine-induced apoptosis

[0086] As expected, neither native PP nor [P 3 PP affected β-cell viability, as assessed by MTT staining ( Figure 1 A). In addition, both peptides inhibited insulin secretion from BRIN-BD11 β-cells induced by 16.7 mM glucose (p<0.05 - p<0.001) and alanine (p<0.05) ( Figure 1 B, Figure 1 C). However, PP and [P 3 PP significantly (p<0.001) enhanced BRIN-BD11 cell proliferation at concentrations of 10 -8 M and 10 -6 M, similar to the manner of GLP-1 ( Figure 1 D). To determine the receptor specificity of PP and [P 3 PP, proliferation experiments were performed in the presence of the specific NPYR4 antagonist (S)-VU0637120 ( Figure 1 D). In the presence of the NPYR4 antagonist, PP and [P 3 PP lacked a significant β-cell proliferative effect ( Figure 1 D). Encouragingly, PP and [P 3 PP also prevented (p<0.05 - p<0.001) cytokine-induced apoptosis of BRIN BD11 β-cells at concentrations of 10 -6 M and 10 -8 M ( Figure 1 E). Similar to the observations on β-cell growth, this protection was abolished by co-incubation with the NPYR4 antagonist (S)-VU0637120 ( Figure 1 E).

[0087] Example 3

[0088] Acute effects of PP peptides on satiety and glucose tolerance in mice

[0089] Intraperitoneal injection of 25 nmol / kg PP or [P 3 PP in overnight-fasted mice resulted in significant (p<0.05 - p<0.01) anorectic effects ( Figure 2 A). However, when administered 4 h before refeeding, only [P 3 PP retained the satiety (p<0.01) effect ( Figure 2 B), highlighting a more prolonged duration of action. When administered to mice in combination with glucose, neither PP nor [P 3 PP had any effect on glucose disposition ( Figure 2 C), while native PP caused a slight decrease (p<0.05) in glucose-stimulated insulin secretion ( Figure 2 D).

[0090] Example 4

[0091] [P 3 PP effects on body weight, food intake, and circulating glucose, insulin, and glucagon in HFF-STZ mice

[0092] In HFF mice with impaired adaptation of STZ-induced β-cells to prolonged high-fat feeding, twice-daily administration of [P 3 PP resulted in significant (p<0.001) and sustained weight loss over a 28-day study period ( Figure 3 A). Weight loss in [P 3 PP-treated HFF-STZ mice began on day 3 and was accompanied by a marked decrease in cumulative energy intake ( Figure 3 B). In addition, by day 7 of [P 3 PP treatment, circulating glucose was significantly decreased (p<0.05), and for the remainder of the study, blood glucose levels in these mice remained relatively normal ( Figure 3 C). Consistent with this, when compared to HFF-STZ saline control mice, [P 3 PP treatment resulted in elevated (p<0.05) terminal plasma insulin concentrations and decreased (p<0.05) glucagon levels ( Figure 3 D, Figure 3 E). This resulted in a significant decrease (p<0.001) in the glucose:insulin ratio in [P 3 PP-treated HFF-STZ mice ( Figure 3 F).

[0093] Example 5

[0094] [P 3 PP effects on glucose tolerance, insulin sensitivity, and pancreatic insulin and glucagon content in HFF-STZ mice

[0095] When compared to saline-treated HFF-STZ control mice, [P 3 PP significantly improved (p<0.001) glucose homeostasis in response to a glucose challenge on day 28 ( Figure 4 A, Figure 4 B). This improved glucose disposal was associated with a significantly amplified (p<0.001) glucose-stimulated insulin secretion response ( Figure 4 C, Figure 4 D). Interestingly, [P 3 PP did not enhance the hypoglycemic effect of exogenous insulin injection in HFF-STZ ( Figure 4 E, Figure 4 F). However, in these [P 3 PP-treated HFF-STZ mice, pancreatic insulin content was elevated (p<0.05) ( Figure 4 G), with no significant effect on pancreatic glucagon ( Figure 4 H).

[0096] Example 6

[0097] [P 3 PP effects on islet morphology, β-cell proliferation, and apoptosis in HFF-STZ mice

[0098] [P 3 PP increased the total islet (p<0.001) area and β-cell (p<0.001) area in HFF-STZ mice ( Figure 5 A). Additionally, treatment with [P 3 PP decreased the area of islet α-cells (p<0.05) ( Figure 5 A).

[0099] In concert with this, [P 3 PP significantly (P<0.001) decreased the α:β ratio in HFF-STZ mice ( Figure 5 B). Treatment with [P 3 PP also decreased the occurrence of glucagon-positive-stained cells within the murine islet core (p<0.05) ( Figure 5 C). In terms of islet size distribution, [P 3 PP intervention decreased (p<0.05) the percentage of smaller-sized islets and increased (p<0.05) the percentage of medium-sized islets ( Figure 5 D). Consistent with the positive [P 3 PP-induced changes in islet structure, in islets treated with [P 3In HFF-STZ treated with [P]PP twice daily for 28 days, β-cell proliferation increased (p<0.001), and the apoptosis rate decreased (p<0.01)( Figure 5 E, Figure 5 F). Representative islet images of insulin and glucagon, insulin and Ki-67, or insulin and TUNEL staining in HFF-STZ mice of each group are shown in Figure 5 G- Figure 5 I, respectively.

[0100] Example 7

[0101] [P 3 PP effects on β-cell lineage in STZ-diabetic Ins1 Cre / + ; Rosa26-eYFP transgenic mice

[0102] In transgenic Ins1 Cre / + ; Rosa26-eYFP mice, diabetes was induced by multiple low-dose STZ injections (50 mg / kg body weight, i.p.) for 5 consecutive days. Then, [P 3 PP (25 nmol / kg) was injected into the mice twice daily for 11 days. The effects on metabolic control, islet morphology, and islet cell lineage were investigated.

[0103] As shown in Figure 6 , [P 3 PP restored food intake and body weight in STZ-diabetic Ins1 Cre / + ; Rosa26-eYFP transgenic mice to near-normal levels, but had no practical effect on blood glucose control.

[0104] As shown in Figure 7 , [P 3 PP significantly reversed the negative effects of STZ on islet morphology, which was associated with an increase in total islet area and β-cell area and a decrease in α-cell area. These positive effects were related to the benefits on islet cell lineage in Ins1 Cre / + ; Rosa26-eYFP transgenic mice ([[]]END Figure 8 ), especially through a reduced loss of β-cell identity (dedifferentiation) and a reduced conversion of adult β-cells to an α-cell-like phenotype (transdifferentiation). In addition, [P 3 PP decreased the α-cell proliferation rate and apoptosis rate, and also decreased β-cell apoptosis and increased proliferation ( Figure 9 and Figure 10 ).

[0105] Example 8

[0106] [P 3 PP effects on STZ-diabetic Glu​CreERT2 ; Effects on α-cell lineage in Rosa26-eYFP transgenic mice

[0107] In transgenic Glu CreERT2 ; Diabetes was induced in Rosa26-eYFP mice by multiple low-dose STZ injections (50 mg / kg body weight, i.p.) for 5 consecutive days. Then, [P 3 PP (25 nmol / kg) was injected twice daily into the mice for 11 days. The effects on metabolic control, islet morphology, and islet cell lineage were studied.

[0108] Compared with Ins1 Cre / + ; Rosa26-eYFP transgenic mice, STZ had a smaller negative impact in Glu CreERT2 ; Rosa26-eYFP mice, but [P 3 PP treatment generally led to a mild improvement in metabolic control ( Figure 11 ). However, [P 3 PP significantly reversed the negative impact of STZ on islet morphology ( Figure 12 ), which was associated with a positive impact on cell lineage ( Figure 13 ). Specifically, this was associated with an increased conversion of GFP+ cells to an insulin-positive phenotype (α-cell transdifferentiation) and an increased dedifferentiation of α-cells ( Figure 13 ).

[0109] Example 9

[0110] Discussion

[0111] The appetite-suppressing effects and beneficial pancreatic endocrine effects of PP and NPYR4 regulation have been previously described. Despite this knowledge, the potential therapeutic utility of PP-based compounds has been largely overlooked. There are many reasons for this. First, PP has a short biological half-life, which limits the use of the natural hormone as a viable therapy. In addition, when compared with the closely related PYY(3-36) peptide hormone, the effect of PP on satiety may be slightly less prominent and is much less characterized. Furthermore, the potential benefits of PP on islet function were not initially very obvious. After acute NPY4R activation in islets, the insulin inhibitory effect dominated, and the more attractive positive effects on β-cell growth and survival were only obvious under more chronic NPYR4 regulation. In fact, a similar phenomenon related to the beneficial effects of NPYR1 activation on islets has only recently been discovered. Therefore, this study aimed to address these issues by generating and expanding the in vitro and in vivo characterization of enzyme-stable PP peptides and further examining the anti-obesity and anti-diabetic benefits in appropriate rodent models.

[0112] Therefore, we utilized the endogenous NPY4R ligand PP and modified the peptide by substituting proline at the 3-position to confer resistance to DPP-4. Such an approach has previously been successfully used to modulate peptide hormones. Importantly, we have preliminarily demonstrated that [P 3 PP retains bioactivity at the pancreatic β-cell level and confers a significant satiety effect in mice, similar to native PP.

[0113] Therefore, [P 3 PP inhibits both glucose-induced insulin release and alanine-induced insulin release, while also promoting β-cell growth and preventing apoptosis. [P 3 PP's β-cell proliferative and anti-apoptotic effects were demonstrated to be dependent on NPY4R activation. Consistent with its limited in vitro insulinotropic effect, when co-injected with glucose into mice, [P 3 PP does not affect glucose homeostasis or circulating plasma insulin. However, PP and [P 3 PP do induce a significant anorectic effect in mice, consistent with the NPYR4-mediated effect on satiety. Additionally, [P 3 PP has an extended duration of biological action in mice, most likely as a result of enhanced enzymatic stability. Collectively, these data suggest that substitution of Leu 3 for Pro 3 in PP confers an extended pharmacodynamic profile without interfering with NPYR4 bioactivity.

[0114] Based on these positive in vitro and in vivo observations, we next examined the beneficial effects of subchronic administration of [P 3 PP in HFF-STZ mice. This mouse model is characterized by STZ-induced blockade of typical β-cell hypertrophy in mice in response to prolonged high-fat feeding, exhibiting obesity and hyperglycemia, making it an ideal model to examine the anti-obesity and anti-diabetic benefits of [P 3 PP. Indeed, twice-daily treatment of these mice with [P 3 PP for 28 days caused a highly significant reduction in energy intake and body weight. Although the weight loss may be a direct reflection of reduced caloric intake, PP has been shown to alter locomotor activity and metabolic rate, which may also be a factor in this regard. Consistent with our findings, PP levels are thought to be reduced in human obesity, and PP infusion results in a significant anorectic effect in obese humans.

[0115] To date, fully translating the anti-obesity benefits of the NPY peptide family into the clinic has been somewhat challenging due to GIT-related side effects, including sweating, nausea, and severe vomiting in humans. However, a sustained-release PYY / NPYR2 analogue formulated with zinc chloride, namely Y14, has recently been described. This formulation shows maintenance of NPYR2 bioactivity but significantly reduces extreme nausea and vomiting in study volunteers. Potentially similar long-acting formulations could be used for other peptide analogues within the NPY family, such as 3 PP. Additionally, PYY(3-36) and PP have been shown to differentially regulate hypothalamic neuronal activity in mice, suggesting that the potential additive effects of these peptides on satiety are worthy of further exploration. Indeed, a dual agonist of NPY2R and NPY4R named obinepitide did progress to clinical trials for obesity, and although it has been discontinued, it still highlights the credible therapeutic promise of this approach.

[0116] In terms of anti-diabetic effects, the circulating glucose concentration in HFF-STZ mice was returned to levels comparable to those observed in normal healthy mice by 3 PP, which was associated with an increase in insulin concentration. Additionally, glucose homeostasis was also significantly improved, which was a direct result of a significant increase in glucose-stimulated insulin secretion. Thus, continuous activation of NPY4R is known to confer significant benefits on overall β-cell function, although short-term NPY4R stimulation has an insulin-suppressive effect. Consistent with this, pancreatic β-cell proliferation was enhanced and apoptosis was reduced by 3 PP treatment in HFF-STZ mice. This ultimately led to an increase in islet area and β-cell area, with fewer small-sized islets and more medium-sized islets, as well as an accompanying increase in pancreatic insulin content. Such positive pancreatic effects are fully consistent with our current in vitro observations of PP and previous studies. Encouragingly, the infiltration of central islet cells with STZ-induced glucagon-positive staining was also essentially reversed by 3 PP. Interestingly, the hypoglycemic effect of exogenous insulin was not significantly enhanced in mice treated with 3 PP, indicating that the beneficial effects on metabolism are mainly related to the improvement of β-cell function rather than insulin action, although the body weight of 3 PP mice was significantly reduced. There was also a decrease in α-cell area and circulating glucagon in HFF-STZ mice treated with 3 PP, consistent with the glucagon-suppressive effect of PP, which was also expected to increase the improved metabolic state.

[0117] In addition, recent attention has turned to the importance of islet cell lineage changes in the development of diabetes and also as potential targets for therapeutic agents. Thus, it is now thought that the loss of β-cell mass in human diabetes is in part related to β-cell dedifferentiation and transdifferentiation into non-insulin-positive islet cell types. Therefore, this also represents an excellent therapeutic target where drugs could help maintain β-cell identity or promote lineage changes of non-β-cells into insulin-positive islet cells, with significant therapeutic potential. We used transgenic mice that allowed tracing of the α-cell lineage and β-cell lineage by using fluorescent tags. We studied in Ins1 Cre / + ; Rosa26-eYFP mice and Glu CreERT2 ; Rosa26-eYFP mice confirmed that important aspects of the antidiabetic action of [P 3 PP at the islet level are related to positive effects on islet cell lineage events. In both transgenic models, [P 3 PP was able to promote the conversion of non-insulin-positive islet cells into an insulin-positive β-cell phenotype. In our rodent diabetes model, these observations provided mechanistic insights into the enhancement of β-cell mass induced by [P 3 PP.

[0118] Despite the apparent benefits of [P 3 PP in the current context, potential limitations need to be considered. Thus, further structural modifications of [P 3 PP at the neprilysin cleavage site, such as Glu 4 -Pro 5 、Ala 22 -Asp 23 or Arg 26 -Tyr 27 , could further improve peptide in vivo stability. However, structure / function studies have shown that alterations at such cleavage sites would come at the cost of a significant reduction in NPY4R affinity. In addition, modifications that limit renal clearance could allow for a more sustained NPY4R activation profile, as demonstrated by related peptide hormone analogues. In addition, the tissue expression profile of NPY4R needs to be fully considered before entering clinical trials. There is good evidence for functional NPYR4 within the CNS, where activation of hypothalamic NPY4R may be [P 3The main mechanism by which PYY induces satiety. In addition, NPY4R regulation within the amygdala is associated with anxiolytic effects and may represent a novel target for treating anxiety-related disorders. Similarly, the stimulation of central GLP-1R is being actively investigated for the treatment of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, as well as for the well-recognized GLP-1-induced anti-obesity benefits. Finally, the need for parenteral delivery of peptide compounds such as PYY can also be considered a potential clinical hurdle. Although small molecule, oral NPY4R agonists have been documented in the literature, the potency, selectivity, and safety of low molecular weight compounds targeting peptide receptors have long been issues regarding therapeutic applicability. In addition, oral delivery of GLP-1 mimetics has now received full clinical approval for diabetes, and oral insulin, calcitonin, parathyroid hormone, and vasopressin have also progressed to clinical trials, indicating that such a delivery route may also be applicable for PYY. 3 The need for PYY can also be considered a potential clinical hurdle. Although small molecule, oral NPY4R agonists have been documented in the literature, the potency, selectivity, and safety of low molecular weight compounds targeting peptide receptors have long been issues regarding therapeutic applicability. In addition, oral delivery of GLP-1 mimetics has now received full clinical approval for diabetes, and oral insulin, calcitonin, parathyroid hormone, and vasopressin have also progressed to clinical trials, indicating that such a delivery route may also be applicable for PYY. 3 PYY.

[0119] In summary, this study demonstrates that enzymatically stable, bioactive, receptor-selective PYY analogs can be generated. In this regard, PYY encompasses the positive β-cell benefits of chronic NPYR4 activation as well as the well-recognized satiety effects of PYY. The significant improvements in islet morphology, insulin secretion, caloric intake, body weight, and overall metabolism induced by PYY in HFF-STZ mice advocate for further preclinical and clinical evaluation of this treatment option for obesity and diabetes. 3 PYY encompasses the positive β-cell benefits of chronic NPYR4 activation as well as the well-recognized satiety effects of PYY. The significant improvements in islet morphology, insulin secretion, caloric intake, body weight, and overall metabolism induced by PYY in HFF-STZ mice advocate for further preclinical and clinical evaluation of this treatment option for obesity and diabetes. 3 PYY in HFF-STZ mice advocate for further preclinical and clinical evaluation of this treatment option for obesity and diabetes.

Claims

1. A peptide analogue of pancreatic polypeptide (PP) for treating diabetes or obesity.

2. The peptide analogue for use according to claim 1, wherein the peptide analogue comprises SEQ ID NO:

1.

3. The peptide analogue for use according to claim 2, wherein the peptide analogue comprises SEQ ID NO:1 and at least one amino acid substitution or modification.

4. The peptide analogue for use according to claim 3, wherein the at least one amino acid substitution comprises leucine substitution for proline.

5. The peptide analogue for use according to claim 4, wherein the peptide analogue comprises SEQ ID NO:

2.

6. A composition for treating diabetes or obesity, wherein the composition comprises SEQ ID NO:1 or SEQ ID NO:2 and a pharmaceutically acceptable carrier.

7. The peptide analogue for use according to any one of claims 1-5, or the composition for use according to claim 6, wherein the use comprises administering a pharmaceutically effective amount of the peptide analogue or the composition.

8. The peptide analogue or composition for use according to claim 7, wherein the use comprises administering 0.25 nmol / kg body weight - 25.00 nmol / kg body weight of the peptide analogue or an equivalent amount of the composition.

9. The peptide analogue for use according to any one of claims 1-8, or the composition for use according to any one of claims 6-8, wherein the diabetes is type 2 diabetes.

10. The peptide analogue for use according to any one of claims 1-9, or the composition for use according to any one of claims 6-9, wherein the use comprises reducing food intake; and / or reducing appetite; and / or reducing blood glucose; and / or increasing insulin levels; and / or improving glucose homeostasis; and / or increasing pancreatic insulin content; and / or increasing total islet area and β-cell area; and / or reducing islet α-cell area; and / or reducing the number of glucagon-positive stained cells; and / or in β-cell dedifferentiation; and / or transdifferentiation into non-insulin-positive islet cell types.

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