Composition for use in treating diabetes or obesity

Abstract

The present invention relates to compositions for use in the treatment of 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 and the use of peptide analogs according to the invention in the manufacture of a medicament for treating diabetes or obesity.

Description

[Technical Field]

[0001] The present invention relates to compositions for use in the treatment of 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 for treating diabetes or obesity, and the use of peptide analogs according to the present invention in the manufacture of a medicament for treating diabetes or obesity. [Background technology]

[0002] The neuropeptide Y (NPY) family of peptides consists of three biologically active peptide hormones: NPY, peptide YY (PYY), and pancreatic polypeptide (PP). These peptides each consist of 36 amino acids and share significant sequence homology. Their biological actions are mediated by binding to and interaction with neuropeptide Y receptors (NPYRs), including NPY1, NPY2, NPY4, and NPY5 receptors. While NPY is thought to play important physiological roles in the central nervous system (CNS), recent research on NPY peptides has highlighted the metabolic benefits of PYY peptides.

[0003] PYY(3-36), an N-terminal cleavage product of dipeptidyl peptidase-4 (DPP-4) from PYY(1-36), is a selective NPY2R agonist with established satiety effects in animals and humans. Initial attempts to exploit the therapeutic benefits of PYY(3-36) and NPYR2 activation in humans failed due to adverse gastrointestinal side effects. However, sustained-release NPY2R agonists have recently been characterized in humans that maintain satiety effects while dramatically reducing side effects, suggesting potential clinical applications. Furthermore, sustained activation of the NPYR1 receptor by native, full-length PYY(1-36) has recently been recognized as beneficial at the pancreatic endocrine level. Thus, prolonged NPY1R activation improves pancreatic β-cell growth, survival, and overall secretory function, as well as aspects of islet cell transdifferentiation. These benefits have been shown to be applicable to isolated human islets in preclinical models of diabetes. Taken together, PYY molecules offer therapeutic potential for anti-obesity 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 for PYY compounds appear to be mutually exclusive.

[0004] An ideal scenario for harnessing the therapeutic effects of NYPR activation would be to encompass both anti-obesity and anti-diabetic effects within the same compound. In this regard, pancreatic polypeptide (PP), which preferentially activates NPY4R, has established satiety effects in both mice and humans. Indeed, the importance of NPY4R signaling in energy homeostasis has been demonstrated by the reduced energy intake and weight gain observed in mice overexpressing PP. Furthermore, treatment of mice with streptozotocin (STZ), a specific β-cell toxin, locally increases PP expression in pancreatic islets, which is thought to occur as an adaptive response to maintain or restore normal islet architecture. Similarly, in vitro observations have revealed that PP exerts a protective effect against STZ-induced DNA damage in pancreatic β cells. Thus, PP and its associated modulation of NPY4R have the potential to integrate the anti-obesity and anti-diabetic effects of NPYR signaling. We must continue our preliminary studies of this compound to further expand on these important observations and advance the potential for novel therapeutic regimens for diabetes.

[0005] Diabetes and obesity are reaching epidemic proportions, and despite the wide range of currently available medications, many people still have poor metabolic control. Therefore, there is a pressing need for new and more effective treatment options. Therefore, there is a clear commercial opportunity for effective antidiabetic and antiobesity drugs with novel mechanisms of action. Summary of the Invention [Means for solving the problem]

[0006] According to a first aspect of the present invention there is provided a peptide analogue of pancreatic polypeptide (PP) for use in the treatment of diabetes or obesity.

[0007] 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 a peptide analogue of pancreatic polypeptide (PP) to a subject in need thereof.

[0008] Optionally, the peptide analog of pancreatic polypeptide (PP) has the amino acid sequence APLEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY.

[0009] Optionally, the peptide analog of pancreatic polypeptide (PP) has an amino acid sequence of APLEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY, as defined by SEQ ID NO:1.

[0010] Optionally, the peptide analog of pancreatic polypeptide (PP) comprises SEQ ID NO: 1 and at least one amino acid substitution or modification.

[0011] Optionally, the peptide analog of pancreatic polypeptide (PP) comprises SEQ ID NO: 1 and at least one amino acid substitution.

[0012] Optionally, the peptide analog of pancreatic polypeptide (PP) comprises SEQ ID NO: 1 and at least one amino acid substitution, wherein the at least one amino acid substitution comprises a leucine to proline substitution.

[0013] Optionally, the peptide analog of pancreatic polypeptide (PP) comprises SEQ ID NO:1 and at least one amino acid substitution, wherein the at least one amino acid substitution comprises a leucine to proline substitution at position 3 of SEQ ID NO:1.

[0014] Optionally, the peptide analog of pancreatic polypeptide (PP) comprises SEQ ID NO: 1 and at least one amino acid substitution, wherein the at least one amino acid substitution comprises a substitution of leucine at position 3 of SEQ ID NO: 1 with proline, and the peptide analog is defined by SEQ ID NO: 2.

[0015] Optionally, the peptide analog of pancreatic polypeptide (PP) has the amino acid sequence APPEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY.

[0016] Optionally, the peptide analog of pancreatic polypeptide (PP) has an amino acid sequence of APPEPVYPGDNAKPEQMAQYAADLRRYINMKTRPRY, as defined by SEQ ID NO:2.

[0017] Preferably, the peptide analog of pancreatic polypeptide (PP) is SEQ ID NO:2.

[0018] According to a third aspect of the present invention there is provided a composition comprising a peptide analogue according to the first or second aspect of the present invention and a pharmaceutically acceptable carrier.

[0019] Optionally, the composition comprises a pharmaceutically effective amount of a peptide analogue according to the first or second aspect of the invention and a pharmaceutically acceptable carrier.

[0020] Optionally, the composition may be administered parenterally.

[0021] Optionally, the composition can be administered parenterally, for example, by injection, such as intramuscular or subcutaneous injection.

[0022] Optionally, the composition may be provided as a liquid composition.

[0023] Optionally, the use comprises administration of a peptide analogue according to the first or second aspect of the invention or a composition according to the third aspect of the invention.

[0024] Further optionally, the use comprises administering to a subject a peptide analogue according to the first or second aspect of the invention or a composition according to the third aspect of the invention.

[0025] Further optionally, the use comprises administering a peptide analogue according to the first or second aspect of the invention or a composition according to the third aspect of the invention to a subject suffering from diabetes.

[0026] Further optionally, the use comprises administering a peptide analogue according to the first or second aspect of the invention or a composition according to the third aspect of the invention to a subject suffering from obesity.

[0027] Optionally, the use comprises administering a pharmaceutically effective amount of a peptide analogue according to the first or second aspect of the invention or a composition according to the third aspect of the invention.

[0028] Further optionally, the use comprises the administration of 0.25 to 25.00 nmol per kg body weight of a peptide analogue according to the first or second aspect of the invention, or an equivalent amount of a composition according to the third aspect of the invention.

[0029] Further optionally, the use comprises the administration of 2.50 to 25.00 nmol per kg body weight of a peptide analogue according to the first or second aspect of the invention, or an equivalent amount of a composition according to the third aspect of the invention.

[0030] Further optionally, the use comprises the administration of 25.00 nmol per kg body weight of a peptide analogue according to the first or second aspect of the invention, or an equivalent amount of a composition according to the third aspect of the invention.

[0031] According to a fourth aspect of the 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 invention or a composition according to the third aspect of the invention.

[0032] According to a fifth aspect of the invention there is provided the use of a peptide analogue according to the first or second aspect of the invention in the manufacture of a medicament for treating diabetes or obesity.

[0033] Optionally, the diabetes is type 2 diabetes.

[0034] Optionally or additionally, the peptide analogue is for reducing food intake. Further optionally or additionally, the peptide analogue is for reducing appetite.

[0035] Optionally, the peptide analogue is for use in lowering blood glucose, and optionally for use in increasing insulin levels.

[0036] Optionally, the peptide analogue is for use in improving glucose homeostasis. Further optionally, the peptide analogue is for use in increasing pancreatic insulin content.

[0037] Optionally, the peptide analogue is for use in increasing total pancreatic islet and beta cell area.

[0038] Optionally, the peptide analogue is for use in reducing pancreatic islet alpha cell area.

[0039] Optionally, the peptide analog is for use in reducing glucagon-positive staining cells.

[0040] Optionally, the peptide analog is for use in beta cell dedifferentiation.

[0041] Optionally, the peptide analog is for use in transdifferentiation into a non-insulin positive pancreatic islet cell type. [Brief explanation of the drawings]

[0042] [Figure 1]Figure 1 shows the effects of PP peptides on cell viability, insulin release, proliferation, and apoptosis of rodent BRIN-BD11 cells. (A) Cells were incubated with test peptides (10-8 to 10-6 M) for 18 hours before adding MTT. (B, C) Cells were incubated (20 minutes) alone or with test peptides (10-12 to 10-6 M) in (B) 16.7 mM glucose or (C) 16.7 mM glucose supplemented with alanine (10 mM), and insulin secretion was assessed by radioimmunoassay (RIA). (D, E) To assess the effects on BRIN BD11 β cell proliferation and apoptosis and receptor selectivity, cells were incubated with test peptides (10-8–10-6 M) alone or with a cytokine mix (IL-1β 100 U / ml, IFN-γ 20 U / ml, TNF-α 200 U / ml) in the presence or absence of the Y4 antagonist (S)-VU0637120 (10-5 M) for 18 h before staining for (D) Ki-67 or (E) TUNEL. Values ​​are means ± SEM (n = 8). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the appropriate control cultures: (A, D, E) medium alone or (B, C) 16.7 mM glucose. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to (C) 10 mM alanine or (E) cytokine mix. Φp<0.05, ΦΦp<0.01, ΦΦΦp<0.001 compared to the effect in the absence of NPYR4 antagonist (S)-VU0637120. [Figure 2]Figure 2 shows the effects of PP peptide on food intake and glucose homeostasis in mice. (A, B) Mice fasted overnight (16 h) were administered the test peptide (25 nmol / kg body weight, i.p.) either 0 h (A) or 4 h (B) before food intake assessment. (C, D) Mice fasted for 16 h were administered the test peptide (25 nmol / kg body weight, i.p.) in combination with glucose (18 mmol / kg body weight, i.p.), and the corresponding AUC values ​​from 0 to 60 min are also shown. Values ​​are means ± SEM from eight mice. *p<0.05, **p<0.01, and ***p<0.001 compared to the appropriate saline (A, B) or glucose-only (C, D) controls. [Figure 3] Figure 3 shows the effects of twice-daily [P3]PP administration for 28 days on (A) body weight change, (B) energy intake, (C) blood glucose levels, and plasma (D) insulin, (E) glucagon, and (F) glucose:insulin ratio in HFF-STZ mice. (A-C) HFF-STZ mice were administered [P3]PP twice daily (25 nmol / kg body weight, i.p.) for 28 days, and parameters were assessed at regular intervals. (D-F) Plasma insulin and glucagon concentrations, as well as the glucose:insulin ratio, were measured on day 28. Values ​​are the mean ± SEM of eight mice. *p<0.05, **p<0.01, ***p<0.001 compared to HFF-STZ controls. [Figure 4]Figure 4 shows the effects of twice-daily [P3]PP administration for 28 days on (A, B) glucose tolerance, (C, D) insulin secretion, (E, F) insulin sensitivity, and (G) insulin and (H) glucagon content in HFF-STZ mice. HFF-STZ mice were treated with [P3]PP (25 nmol / kg body weight, i.p.) twice daily for 28 days, and all parameters were assessed. (A, B) Blood glucose levels and (C, D) plasma insulin were measured before and after administration of glucose alone (18 mmol / kg, i.p.) at t = 0 min in overnight-fasted mice. (E, F) Blood glucose levels were assessed in non-fasted mice after administration of insulin (15 U / kg body weight, i.p.) at t = 0 min. (G, H) Pancreatic insulin and glucagon content were measured by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), respectively. Values ​​are the mean ± SEM of eight mice. *p<0.05, **p<0.01, ***p<0.001 compared to HFF-STZ control. [Figure 5] Figure 5 shows the effect of twice-daily [P3]PP treatment for 28 days on islet morphology in HFF-STZ mice. After 28 days of twice-daily administration of [P3]PP (25 nmol / kg body weight, i.p.), HFF-STZ mice were treated with [P3]PP. (A) Area of ​​islets, β cells, and α cells, (B) α:β ratio, (C) islet size distribution, (D) percentage of centrally stained cells positive for glucagon, and (E) proliferation and (F) apoptosis of β cells were assessed. Islet morphology was assessed using CellF image analysis software, and β cell proliferation and apoptosis were measured by Ki-67 staining or TUNEL staining, respectively. (G-I) Representative islet images (40x 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. Values ​​are the mean ± SEM of eight mice. *p<0.05, **p<0.01, ***p<0.001 compared to HFF-STZ control. [Figure 6]Figure 6 shows the effects of twice-daily [P3]PP (25 nmol / kg body weight, ip administration) on body weight, energy intake, and circulating glucose in streptozotocin (STZ)-induced diabetic Ins1Cre / +;Rosa26-eYFP mice. (A) Body weight, (B) % body weight change, (C) cumulative calorie expenditure, (D) blood glucose level, (E) non-fasting blood glucose level, and (F) fasting blood glucose level. Values ​​are means ± SEM of seven mice. *P<0.05, **P<0.01, ***P<0.001 compared to STZ-diabetic controls. [Figure 7] Figure 7 shows the effect of twice-daily [P3]PP (25 nmol / kg body weight) on pancreatic morphology in STZ Ins1Cre / +;Rosa26-eYFP mice. (A-D) (A) Pancreatic islet, (B) beta, (C) alpha cell area, and (D) alpha:beta ratio were measured using CellF image analysis software. (E) Representative images (40x magnification) of pancreatic islets showing insulin (red), glucagon (green), and DAPI (blue) immunoreactivity. Values ​​are means ± SEM from seven mice. **P<0.01 and ***P<0.001 compared to STZ control. [Figure 8] Figure 8 shows the effect of twice-daily [P3]PP (25 nmol / kg body weight) on pancreatic β-cell identity in STZ Ins1Cre / +;Rosa26-eYFP mice. (A-D) (A) β-cell dedifferentiation (insulin-positive, GFP+ positive cells) and (B) β-to-α transdifferentiation (glucagon-positive, GFP+ cells) were measured using CellF image analysis software. Representative images (40x magnification) of pancreatic islets from each group of mice showing immunoreactivity for (C) insulin (red) or (D) glucagon (red) with GFP (green) and DAPI (blue). Values ​​are the mean ± SEM of seven mice. **P<.01 and ***P<.001 compared to STZ control. [Figure 9]Figure 9 shows the effect of twice-daily [P3]PP pancreatic polypeptide (25 nmol / kg body weight) on pancreatic α-cell turnover rate in STZ Ins1Cre / +;Rosa26-eYFP mice. (A) Quantification of α-cell proliferation and (B) α-cell apoptosis rate. Representative images (40x magnification) of pancreatic islets showing (C) glucagon (green), Ki-67 (red), and DAPI (blue) or (D) glucagon (red) and TUNEL staining (green); scale bar 100 μm. Values ​​are means ± SEM of six islets per group. Approximately 50 islets were analyzed per group. *p<0.05, **p<0.01, ***p<0.001 compared to matched non-diabetic controls. Δp<0.05, ΔΔp<0.01 compared to matched STZ controls. [Figure 10] Figure 10 shows the effect of twice-daily [P3]PP pancreatic polypeptide (25 nmol / kg body weight) on pancreatic β-cell turnover in STZ Ins1Cre / +;Rosa26-eYFP mice. A) Quantification of β-cell proliferation and (B) β-cell apoptosis rates. Representative images (40x magnification) of islets showing (C) insulin (green), Ki-67 (red), and DAPI (blue), or (D) insulin (red) and TUNEL staining (green); scale bar 100 μm. Values ​​are means ± SEM from six mice per group, with approximately 50 islets analyzed per group. ***p<0.001 compared to matched nondiabetic controls. ΔΔΔp<0.001 compared to matched STZ controls. [Figure 11] Figure 11 shows the effects of twice-daily administration of [P3]PP (25 nmol / kg body weight, i.p.) on body weight, energy intake, and circulating glucose in streptozotocin (STZ)-induced diabetic GluCreERT2;Rosa26-eYFP mice. (A) Body weight, (B) % body weight change, (C) cumulative calorie expenditure, (D) blood glucose level, (E) non-fasting blood glucose level, and (F) fasting blood glucose level. Values ​​are means ± SEM of seven mice. *P<0.05, **P<0.01, and ***P<0.001 compared to STZ-diabetic controls. [Figure 12]Figure 12 shows the effect of twice-daily [P3]PP (25 nmol / kg body weight) on pancreatic morphology in STZ GluCreERT2;Rosa26-eYFP mice. (A-D) (A) Pancreatic islet, (B) beta, (C) alpha cell area and (D) alpha:beta ratio were measured using CellF image analysis software. (E) Representative images (40x magnification) of islets from each group of mice showing insulin (red), glucagon (green), and DAPI (blue) immunoreactivity. Values ​​are means ± SEM of seven mice. *P<0.05, **P<0.01, ***P<0.001 compared to STZ control. [Figure 13] Figure 13 shows the effect of twice-daily [P3]PP (25 nmol / kg body weight) on pancreatic α-cell identity in STZ GluCreERT2;Rosa26-eYFP mice. (A-D) (A) α-cell transdifferentiation (insulin+ve, GFP+ve cells) and (B) α-cell dedifferentiation (glucagon+ve, GFP+ cells) were measured using CellF image analysis software. (C) Representative images (40x magnification) of pancreatic islets from each group of mice showing immunoreactivity for insulin (red) or glucagon (red) with GFP (green) and DAPI (blue). Values ​​are means ± SEM of seven mice. **p<0.01 compared to appropriate non-diabetic controls. Δp<0.05, ΔΔp<0.01 compared to appropriate STZ controls. DETAILED DESCRIPTION OF THE INVENTION

[0043] Materials and Methods peptide PP peptides were synthesized by Synpeptide (Shanghai, China) with 95% purity and confirmed in-house 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, MA, USA) and subsequently eluted on a Kinetex C-18 analytical column (150 x 4.60 mm, Phenomenex, Cheshire, UK) 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 over 240 min. The column effluent was monitored by UV absorbance at 214 nm. For peptide mass detection, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Perspective Biosystems, USA) was used in positive detection mode with α-cyano-4-hydroxycinnamic acid as the matrix.

[0044] Enzymatic Stability PP and [P 3 To establish the in vitro stability of ]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 tested using RP-HPLC and MALDI-ToF using the same system as above.

[0045] In vitro effects on insulin secretion, cell viability, receptor selectivity, and β-cell proliferation and survival Preliminary studies have demonstrated the effects of natural PP and [P] on glucose-induced insulin secretion, β-cell proliferation, cell viability, and survival in BRIN-BD11 cells. 3The effects of PP on insulin secretion were investigated. Cells were cultured in RPMI 1640 medium (Gibco Life Technologies Ltd) supplemented with 10% v / v fetal bovine serum (Gibco) and 1% v / v antibiotics (0.1 mg / ml streptomycin and 100 U / ml penicillin) at 37°C in 5% CO2. For insulin secretion experiments, cells were seeded at a density of 150,000 cells per well in 24-well plates (Falcon Ltd). After overnight fixation, the medium was removed, and cells were preincubated with 1.1 mM glucose KRB buffer for 40 min. Next, the preincubation buffer was removed, and cells were preincubated with 16.7 mM glucose and PP test peptide (10 -12 ~10 -6 In a second series of experiments, 1 ml of KRB test solution containing 16.7 mM glucose and 10 mM alanine was added after incubation with the PP test peptide (10 -8 ~10 -6 The insulinotropic effect of α-glucan (M) was measured. In all experiments, after a 20-minute incubation, supernatants were collected and stored at -20°C until measurement of insulin concentrations by a fully characterized dextran-coated charcoal radioimmunoassay (Flatt and Bailey, 1981).

[0046] To evaluate the effect of PP peptides on β-cell viability, BRIN-BD11 cells (40,000 cells / well) were incubated with test peptides (10 -6 and 10 -8 After 18 hours of incubation with the test peptide (10 M), the MTT assay was performed as previously described (Green et al., 2016). Briefly, cells were seeded in 96-well plates at a density of 10,000 cells per well in RMPI-1640 medium and incubated in a CO2 incubator with the test peptide (10 -8 ~10 -6The cells were incubated for 24 hours in the presence or absence of ATP. After incubation, 20 μl of MTT solution (5 mg / ml) was added to the cells and incubated at 37°C for 2 hours. The medium was then removed, and the formazan crystals were dissolved with 100 μl of DMSO while agitating the plate for 10 minutes. The absorbance was read on a spectrophotometer at an excitation wavelength of 570 nm and an emission wavelength of 630 nm.

[0047] PP peptide (10) on β-cell proliferation and protection from apoptosis -8 and 10 -6 To examine the effect of GLP-1 (10 M), BRIN-BD11 cells were seeded onto sterile clear glass cover slips (16 mm diameter) at a density of 40,000 cells per well in 12-well plates (Falcon Ltd) and cultured for 18 hours. -8 and 10 -6M), and a cytokine cocktail mix (IL-1β (100 U / mL), IFNγ (20 U / mL), and TNFα (200 U / mL)) were used as appropriate controls. Cells were then washed with PBS and fixed with 4% paraformaldehyde. Antigen retrieval was performed with sodium citrate buffer for 20 minutes at 95°C, followed by blocking with 2% BSA for 45 minutes. For proliferation studies, coverslips were incubated with rabbit anti-Ki-67 primary antibody (Abcam, ab15580) at 37°C, followed by Alexa Fluor® 488 secondary antibody. Coverslips were mounted on polylysine-coated microscope slides using a 50:50 glycerol:PBS solution and stored at 4°C until required for analysis. To determine the protective ability of the PP peptide against cytokine-induced apoptosis, BRIN BD11 cells were seeded, washed, and fixed as previously described, except that the cytokine cocktail was added to the culture medium. The coverslips were then incubated with TUNEL reaction mix (Roche Diagnostics Ltd, UK) for 60 min at 37°C and mounted on microscope slides as previously described. All slides were observed under a fluorescence microscope (Olympus System Microscope, model BX51, Southend-on-Sea, UK) and photographed with a DP70 camera adapter system. The proliferation / TUNEL-positive frequency was determined using the cell-counter function of ImageJ Software and expressed as a percentage of the total cells analyzed, as previously described in our laboratory. In another series, specific Y4 receptor antagonists (10 -5 M, (S)-VU0637120, Glixx Laboratories Inc.) was used to evaluate the receptor selectivity of the PP peptides related to β-cell proliferation and anti-apoptotic effects.

[0048] Acute in vivo experiments Experiments were performed using 12-week-old adult Swiss mice (Envigo Ltd, UK). Mice were housed in a temperature-controlled unit environment (22 ± 2°C) under a 12-h light / dark cycle and had free access to drinking water and a maintenance diet (10% fat, 30% protein, 60% carbohydrate; Trouw Nutrition, UK). Mice (n = 8) were fasted overnight (16 h) and then intraperitoneally (i.p.) injected with glucose alone (18 mmol / kg body weight) or in combination with the test peptide (25 nmol / kg body weight). The acute effects of the PP test peptide on glucose homeostasis and insulin secretion were assessed. To assess the effect of the PP peptide on food intake, mice (n = 8) were fasted for 16 h and then i.p. injected with saline vehicle (0.9% [w / v] NaCl) or the test peptide (25 nmol / kg body weight), and cumulative food intake was monitored over 150 min. Long-lasting effects on feeding were examined by injecting peptide (25 nmol / kg body weight) or saline 4 hours before feeding.

[0049] Chronic in vivo experiments In a preliminary experiment, male NIH Swiss adult mice (12 weeks old) were maintained on a high-fat diet (45% fat, 20% protein, 35% carbohydrate, total energy ratio 26.15 kJ / g, Dietex International Ltd., Witham, UK) for 3 weeks. After this period, streptozotocin (50 mg / kg body weight, dissolved in sodium citrate buffer, pH 4.5, after a 4-hour fast) was injected intravenously three times weekly for weeks 3, 4, and 5. From week 6, diabetic mice (non-fasting blood glucose >11.1 mmol / L) were divided into groups (n = 8) and administered either saline vehicle (0.9% [w / v] NaCl) or [P 3 ]PP (25 nmol / kg body weight) was intraperitoneally injected twice daily (8:00 AM and 8:00 PM) for 28 days. Mice were maintained on a high-fat diet throughout the experiment. Transgenic mice, i.e., Ins1Cre / +;Rosa26-eYFP and Glu CreERT2In a study using Rosa26-eYFP mice, diabetes was induced by multiple low-dose streptozotocin (STZ) injections (4-hour fasting, 50 mg / kg body weight, i.p., sodium citrate buffer, pH 4.5) for 5 consecutive days. Once the onset of diabetes was biochemically confirmed, the mice were treated twice daily (9:00 AM and 5:00 PM) with saline (0.9% (w / v) NaCl) or [P 3 ]PP (25 nmol / kg body weight) was administered for 11 days. In all experiments, cumulative energy intake and body weight were assessed at regular intervals, and circulating glucose, insulin, and glucagon were measured at the end of the treatment period, as appropriate. In addition, glucose tolerance tests (18 mmol / kg body weight, administered i.p., 18-h fasting) and insulin sensitivity tests (15 U / kg bovine insulin, administered i.p., non-fasting) were performed as needed. For final analysis, pancreatic tissue was dissected and processed for protein extraction with acid ethanol or fixation in 4% PFA, respectively, for quantification of hormone content or islet morphology. All animal experiments were approved by the Ulster University Animal Ethics Review Committee and conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.

[0050] biochemical analysis Blood samples were collected from conscious mice via an incision at the tip of the tail vein, and blood glucose levels were immediately measured using an Ascencia Contour blood glucose meter (Bayer Healthcare Newbury, UK). Blood was collected into chilled heparin / fluoride-coated microcentrifuge tubes (Sarstedt, Nümbrecht, Germany) and centrifuged at 12,000 rpm for 15 minutes in a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) to separate plasma. Insulin and glucagon levels were then measured by in-house radioimmunoassay (Flatt and Bailey, 1981) or a commercially available ELISA (EZGLU-30K, Merck Millipore, Burlington, MA), respectively.

[0051] immunohistochemistry Islet morphology was examined by immunohistochemical staining for insulin (1:400, ab6995, AbCam) or glucagon (1:1000, ab92517, AbCam). Image analysis was performed using CellF image analysis software (Olympus Soft Imaging Solutions, GmbH, Münster, Germany). To assess islet morphology, the areas of insulin- and glucagon-positive staining were quantified using the "closed polygon" available in CellF image analysis software, and the islet / β- / α-cell area (μm) was calculated. 2The total number of islets was expressed as a function of the total number of islets in the β-cell population. β-cell proliferation and apoptosis were examined by co-staining with insulin and Ki-67 (1:400; ab15580, AbCam) or TUNEL (in situ cell death kit, Fluorescein, Roche Diagnostics). For quantification, the number of insulin-positive cells co-expressing Ki-67 or TUNEL, respectively, was counted using ImageJ software. At least 80 islets per treatment group were analyzed. For transdifferentiation studies in transgenic mice, co-staining with insulin or glucagon and GFP (1:400, ab5450, AbCam) was used as previously described. In all cases, after incubation with the primary antibody, secondary antibodies, Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit (1:400, ThermoFisher Scientific), were used as appropriate. Slides were given a final incubation in DAPI before imaging 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.

[0052] statistical analysis Statistical tests were performed using GraphPad PRISM software (version 5.0). Values ​​are expressed as mean ± SEM. Comparisons between groups were performed using one-way analysis of variance (with Bonferroni post hoc test), two-way analysis of variance (with Bonferroni post hoc test), or Student's unpaired t-test, as appropriate. Differences were considered significant when p<0.05. [Example]

[0053] Embodiments of the present invention will now be illustrated by way of non-limiting examples.

[0054] Example 1 In vitro DPP-4 stability As expected, incubation of PP with DPP-4 produced the N-terminally cleaved product PP(3-36) (Table 1). 3 ]PP was completely resistant to degradation by DPP-4 (Table 1).

[0055] [Table 1]

[0056] Natural PP and [P using one-letter amino acid abbreviations 3 ]PP. 3 Amino acid substitutions in PP are underlined and in bold. In vitro DPP-4 stability was assessed after incubating test peptides with purified DPP-4 (5 mU) for 0–8 h.

[0057] Example 2 Effects of PP peptides on cell viability, insulin release, receptor selectivity, β-cell proliferation, and protection from cytokine-induced apoptosis As expected, the native PP and [P 3 Neither PP nor [P]PP affected β-cell viability (Fig. 1A). 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) (Fig. 1B, C). On the other hand, PP and [P 3 ]PP, like GLP-1, -8 and 10 -6 At a concentration of M, PP and [P 3 To determine the receptor specificity of ]PP, we performed proliferation experiments in the presence of the specific NPYR4 antagonist (S)-VU0637120 (Fig. 1D). 3 ]PP did not show any obvious β-cell proliferation effects (Fig. 1D). 3] PP is 10 -6 M and 10 -8 At concentrations of 1000 M, it also protected against cytokine-induced apoptosis in BRIN BD11 β cells (p<0.05–p<0.001) (Figure 1E). Similar to the observations regarding β-cell proliferation, this protective effect was abolished by co-incubation with the NPYR4 antagonist (S)-VU0637120 (Figure 1E).

[0058] Example 3 Acute effects of PP peptide on satiety and glucose tolerance in mice Mice were fasted overnight and given 25 nmol / kg of PP or [P 3 When PP was intraperitoneally injected, a significant (p<0.05-p<0.01) appetite suppression effect was observed (Figure 2A). However, when PP was administered 4 hours before refeeding, [P 3 Only PP maintained its satiating effect (p<0.01) (Fig. 2B), highlighting its prolonged duration of action. When administered in combination with glucose to mice, PP and [P 3 ] PP had no effect on glucose disposal (Fig. 2C), whereas native PP caused a slight decrease (p<0.05) in glucose-stimulated insulin secretion (Fig. 2D).

[0059] Example 4 [P] on body weight, food intake, and circulating glucose, insulin, and glucagon in HFF-STZ mice 3 ] PP effect In HFF mice, where long-term high-fat diet feeding reduced STZ-induced β-cell adaptation, [P 3 ]PP administered twice daily resulted in significant weight loss (p<0.001) that persisted throughout the 28-day study period (Fig. 3A). 3 The weight loss in PP-treated HFF-STZ mice was accompanied by a clear decrease in cumulative energy intake from day 3 onwards (Fig. 3B). 3]PP treatment significantly reduced circulating glucose on day 7 (p<0.05), and these mice maintained relatively normal blood glucose levels for the remainder of the study (Figure 3C). Similarly, [P 3 ]PP treatment increased peripheral plasma insulin levels (p<0.05) and decreased glucagon levels (p<0.05) (Fig. 3D, E). 3 ] The glucose:insulin ratio was clearly reduced (p<0.001) in PP-treated HFF-STZ mice (Fig. 3F).

[0060] Example 5 [P] on glucose tolerance, insulin sensitivity, and pancreatic insulin and glucagon contents in HFF-STZ mice 3 ] PP effect [P 3 ]PP significantly improved glucose homeostasis in response to a glucose load on day 28 compared with saline-treated HFF-STZ control mice (p<0.001) (Fig. 4A, B). This improvement in glucose disposal was associated with a significant amplification of the glucose-stimulated insulin secretory response (p<0.001) (Fig. 4C, D). Interestingly, [P 3 ]PP did not enhance the glucose-lowering effect of exogenous insulin injection in HFF-STZ (Fig. 4E, F). However, these [P 3 ] In PP-treated HFF-STZ mice, pancreatic insulin content was elevated (p<0.05) (Fig. 4G), with no obvious effect on pancreatic glucagon (Fig. 4H).

[0061] Example 6 [P] on islet morphology, β-cell proliferation, and apoptosis in HFF-STZ mice 3 ] PP effect [P 3 ]PP increased total islet area (p<0.001) and β-cell area (p<0.001) in HFF-STZ mice (Figure 5A). Furthermore, islet α-cell area increased significantly with [P 3] was reduced by PP treatment (p<0.05) (Fig. 5A).

[0062] In harmony with this, [P 3 ]PP significantly (P<0.001) reduced the α:β ratio in HFF-STZ mice (Fig. 5B). The appearance of glucagon-positive cells in the islet core of mice was also significantly reduced by [P 3 ] was reduced by PP treatment (p<0.05) (Fig. 5C). 3 Treatment with PP decreased the proportion of small islets (p<0.05) and increased the proportion of medium-sized islets (p<0.05) (Figure 5D). 3 ] Similar to the favorable changes induced by PP, [P 3 HFF-STZ mice treated with PP twice daily for 28 days showed enhanced β-cell proliferation (p<0.001) and reduced apoptosis (p<0.01) (Figure 5E, F). Representative images of islets from each group of HFF-STZ mice stained for insulin and glucagon, insulin and Ki-67, or insulin and TUNEL are shown in Figure 5G-I, respectively.

[0063] Example 7 STZ Diabetes Ins1 Cre / + [P] for the β-cell lineage in Rosa26-eYFP transgenic mice 3 ] PP effect Transgenic Ins1 Cre / + Diabetes was induced in Rosa26-eYFP mice by multiple low-dose STZ injections (50 mg / kg body weight, i.p.) for 5 consecutive days. 3 ]PP (25 nmol / kg) was injected twice daily for 11 days, and the effects on metabolic control, islet morphology, and islet cell lineage were investigated.

[0064] As shown in Figure 6, [P 3 ]PP is STZ diabetes Ins1 Cre / +In Rosa26-eYFP transgenic mice, food intake and body weight were restored to near-normal levels, but glycemic control was not substantially affected.

[0065] As shown in Figure 7, [P 3 PP significantly ameliorated the negative effects of STZ on islet morphology, which was associated with an increase in total islet and β-cell area and a decrease in α-cell area. These favorable effects were due to the Ins1 Cre / + This was associated with benefits to the islet cell lineage in Rosa26-eYFP transgenic mice (Figure 8), specifically through reduced loss of β-cell identity (dedifferentiation) and reduced conversion of adult β-cells to an α-cell-like phenotype (transdifferentiation). Additionally, [P 3 ]PP decreased the proliferation and apoptosis rate of α-cells, and simultaneously decreased apoptosis and increased proliferation of β-cells (Figs. 9 and 10).

[0066] Example 8 STZ Diabetes Glu CreERT2 ;[P 3 ] PP effect 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. 3 ]PP (25 nmol / kg) was injected twice daily for 11 days, and its effects on metabolic regulation, islet morphology, and islet cell lineage were investigated.

[0067] STZ is Ins1 Cre / + Glu compared with Rosa26-eYFP transgenic mice CreERT2 ;Rosa26-eYFP mice showed less negative effects, but [P 3 ]PP treatment generally resulted in a modest improvement in metabolic control (Fig. 11).3 PP significantly ameliorated the negative effects of STZ on islet morphology (Figure 12), which was associated with favorable effects on cell lineages (Figure 13). Specifically, this was associated with increased conversion of GFP+ cells to an insulin-positive phenotype (alpha-cell transdifferentiation) and increased alpha-cell dedifferentiation (Figure 13).

[0068] Example 9 Consideration The appetite suppressant and beneficial pancreatic endocrine effects of PP and NPYR4 modulation have been reported. Despite these findings, the potential therapeutic utility of PP-based compounds has largely been overlooked. This is due to a variety of reasons. First, PP's short biological half-life limits the use of this natural hormone as an effective therapeutic agent. Additionally, PP's effects on satiety are less pronounced and less well characterized compared to the closely related PYY(3-36) peptide hormone. Furthermore, the potential benefits of PP on pancreatic islet function were initially unclear; only the insulinostatic actions that prevail after acute NPY4R activation in pancreatic islets and the more compelling favorable effects on β-cell growth and survival were evident with more chronic NPYR4 modulation. Indeed, a similar phenomenon has only recently been identified for the beneficial effects of NPYR1 activation in pancreatic islets. In this study, we aimed to address these issues by generating an enzymatically stable PP peptide, extensively characterizing it in vitro and in vivo, and further examining its anti-obesity and anti-diabetic effects in appropriate rodent models.

[0069] Therefore, we took the endogenous NPY4R ligand, PP, and modified the peptide by substituting proline at position 3 to confer resistance to DPP-4. This approach has been successfully employed previously with regulatory peptide hormones. Importantly, [P3 ] It was confirmed for the first time that PP retains its physiological activity at the pancreatic beta cell level and exerts a clear satiety effect in mice similar to that of native PP.

[0070] In this way, [P 3 ]PP inhibited both glucose- and alanine-induced insulin secretion, while promoting β-cell proliferation and protecting them from apoptosis. [P 3 The beta cell proliferation and anti-apoptotic effects of PP were confirmed to be dependent on NPY4R activation. 3 ]PP did not affect glucose homeostasis or circulating plasma insulin when co-injected with glucose in mice. However, PP and [P 3 ]PP induced a clear appetite suppression effect in mice, consistent with an NPYR4-mediated effect on satiety. 3 ]PP prolonged the duration of biological action in mice, likely as a result of its enhanced enzymatic stability. Overall, these data support the enhanced potency of Leu in PP. 3 Pro 3 These results demonstrate that substitution with confers a long-lasting pharmacodynamic profile without interfering with the biological activity of NPYR4.

[0071] Based on these favorable in vitro and in vivo observations, we next investigated the effects of [P 3 This mouse model is characterized by the inhibition of STZ-induced classical β-cell hypertrophy and the development of both obesity and hyperglycemia after long-term high-fat diet feeding. 3 ] is an ideal model for investigating the anti-obesity and anti-diabetic effects of PP. 3] Administration of PP twice daily for 28 days induced highly significant reductions in energy intake and body weight in these mice. While weight loss likely reflects a direct reduction in caloric intake, PP has been shown to alter locomotor activity and metabolic rate, which may also contribute. Consistent with our findings, PP levels are thought to be reduced in obese humans, and PP infusion has a pronounced appetite-reducing effect in obese humans.

[0072] Translating the full anti-obesity benefits of the NPY peptide family into clinical practice has been somewhat difficult in humans due to gastrointestinal (GIT)-related side effects, such as sweating, nausea, and severe vomiting. However, a sustained-release PYY / NPYR2 analogue formulated with zinc chloride, namely Y14, has recently been reported. This formulation has been shown to maintain NPYR2 biological activity while significantly reducing severe nausea and vomiting in test volunteers. [P 3 Similar long-acting formulations may be used for other peptide analogs of the NPY family, such as PP. Furthermore, PYY(3-36) and PP have been shown to differentially regulate hypothalamic neuronal activity in mice, suggesting a potential additive effect of these peptides on satiety, which merits further testing. In fact, a dual NPY2R and NPY4R agonist called obinepitide advanced into clinical trials for obesity, but was discontinued, demonstrating therapeutic promise for this approach.

[0073] Regarding the antidiabetic effect, the circulating glucose concentration in HFF-STZ mice was [P 3]PP restored insulin levels to levels similar to those observed in normal healthy mice, which was linked to elevated insulin concentrations. In addition, glucose homeostasis was significantly improved as a direct result of the large increase in glucose-stimulated insulin secretion. Thus, sustained activation of NPY4R is known to have profound effects on overall β-cell function, even if there are insulinostatic effects of short-term NPY4R stimulation. Accordingly, we administered [P 3 Administration of ]PP enhanced pancreatic β-cell proliferation and reduced apoptosis. This resulted in increased islet and β-cell area, a decrease in small islets, an increase in medium-sized islets, and an increase in pancreatic insulin production. These beneficial effects on the pancreas are fully consistent with our in vitro observations and previous studies using PP. Encouragingly, STZ-induced infiltration of glucagon-positive central islet cells was also suppressed by [P 3 ]PP substantially improved the 3 In mice treated with ]PP, the hypoglycemic effect of exogenous insulin was not dramatically enhanced, which is due to the 3 Despite the significant weight loss in PP mice, this suggests that the metabolic benefits are primarily linked to improved β-cell function rather than insulin action. 3 ] In HFF-STZ mice treated with PP, α-cell area and circulating glucagon were reduced, similar to the glucagonostatic actions of PP, which are also expected to contribute to improved metabolic status.

[0074] In addition, recent attention has focused on the importance of islet cell lineage changes in the development of diabetes, and their potential as a therapeutic target. Therefore, it is now believed that the loss of beta cell mass in human diabetes is partly due to not only beta cell dedifferentiation but also transdifferentiation into non-insulin-positive islet cell types. Consequently, beta cell mass loss may also be an excellent therapeutic target, and drugs that help preserve beta cell identity or promote the lineage conversion of non-beta cells into insulin-positive islet cells have clear therapeutic potential. We used transgenic mice that can track alpha and beta cell lineages using fluorescent tags. Ins1Cre / +;Rosa26-eYFP mice and Glu CreERT2 Studies using Rosa26-eYFP mice have shown that [P 3 It was confirmed that an important aspect of the antidiabetic effect of PP is related to its beneficial effects on islet cell lineage events. 3 ]PP was able to promote the conversion of insulin-negative islet cells to an insulin-positive β-cell phenotype. These observations are consistent with the efficacy of [P 3 ]This provides insight into the mechanism by which PP increases beta cell mass.

[0075] In the current situation [P 3 While the advantages of PP are clear, potential limitations must also be considered. 4 -Pro 5 , Ala 22 -Asp 23 Or Arg 26 -Tyr 27 [P] at the neprilysin cleavage site, such as 3Further structural modifications of ]PP may further improve the stability of the peptide in vivo. However, structural / functional studies suggest that these cleavage site changes come at the cost of significantly reducing NPY4R affinity. Furthermore, modifications that limit renal clearance may enable a more sustained NPY4R activation profile, as demonstrated with related peptide hormone analogs. Furthermore, the tissue expression profile of NPY4R also needs to be thoroughly explored before proceeding to the clinic. There is ample evidence demonstrating the presence of functional NPYR4 in the CNS, and activation of the hypothalamic NPY4R is associated with [P 3 ] is thought to be the primary mechanism for satiety induction by [P]. Furthermore, NPY4R modulation in the amygdala has been linked to anxiolytic effects and may represent a novel target for treating anxiety-related disorders. Similarly, stimulation of central GLP-1R is actively being investigated as a therapeutic agent for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and is also well known to be the primary mechanism for the anti-obesity effects of GLP-1. Finally, [P] 3 The need for parenteral delivery of peptide compounds such as NPY4R agonists has also been identified as a potential barrier to clinical application. Although orally available, small molecule NPY4R agonists have been described in the literature, the efficacy, selectivity, and safety of small molecule compounds targeting peptide receptors have long been questionable in terms of their therapeutic applicability. Furthermore, oral delivery of GLP-1 mimetics has now received full clinical approval for diabetes, and orally available insulin, calcitonin, parathyroid hormone, and vasopressin are also progressing in clinical trials, making the potential for clinical application of NPY4R agonists such as NPY4R agonists and NPY4R agonists likely to be more promising. 3 ]This suggests that this delivery route may also be effective for PP.

[0076] In conclusion, this study demonstrates that enzymatically stable, bioactive, and receptor-selective PP analogs can be generated. 3 In addition to the recognized satiety effects of PP, PP also contains positive benefits to β cells through chronic NPYR4 activation.3 ]PP significantly improved islet morphology, insulin secretion, caloric intake, body weight, and overall metabolism, suggesting further preclinical and clinical evaluation of this treatment option for obesity and diabetes.

Claims

1. A peptide analogue of pancreatic polypeptide (PP) for use in the treatment of diabetes or obesity.

2. The peptide analog of claim 1 , wherein the peptide analog comprises SEQ ID NO:

1.

3. 3. The peptide analog of claim 2, wherein the peptide analog comprises SEQ ID NO: 1 and at least one amino acid substitution or modification.

4. 4. The peptide analog of claim 3, wherein the at least one amino acid substitution comprises a leucine to proline substitution.

5. The peptide analog of claim 4, wherein the peptide analog comprises SEQ ID NO:

2.

6. A composition for use in treating diabetes or obesity, said composition comprising SEQ ID NO: 1 or SEQ ID NO: 2 and a pharmaceutically acceptable carrier.

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

8. 8. The peptide analogue or composition of claim 7, wherein said use comprises administering 0.25 to 25.00 nmol of the peptide analogue or equivalent amount of the composition per kg of body weight.

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

10. 10. The peptide analogue of any one of claims 1 to 9 or the composition of any one of claims 6 to 9, wherein said 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 pancreatic islet and beta cell area and / or decreasing pancreatic islet alpha cell area and / or decreasing glucagon positive staining cells and / or dedifferentiation of beta cells and / or transdifferentiation into non-insulin positive pancreatic islet cell types.

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