A composition of polypeptide analogs of GLP-1 and PYY 3-36 and methods of making and using the same

Abstract

The application discloses a composition of GLP-1 and PYY 3‑36 polypeptide analogues and a preparation method and application thereof, and relates to the technical field of biological medicines. The composition comprises: a GLP-1 polypeptide analogue and a PYY 3‑36 polypeptide analogue. Compared with GLP-1 natural sequences and PYY 3‑36 natural sequences, the composition of the GLP-1 polypeptide analogue and the PYY 3‑36 polypeptide analogue has remarkable effects on improving metabolic diseases such as obesity, diabetes and fatty liver and emotional diseases such as anxiety and depression.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a GLP-1 and PYY 3-36 Compositions of polypeptide analogs, their preparation methods, and applications. Background Technology

[0002] Obesity increases the risk of diseases such as diabetes mellitus (DM) and fatty liver disease (FL). The World Health Organization defines obesity as a chronic disease characterized by an excess or abnormality of adipose tissue due to disordered regulation of normal body fat mass. Its development and severity are influenced by both endogenous and exogenous (environmental) factors. [1, 2] Diabetes mellitus (DM) is a serious chronic metabolic disorder caused by the interaction of genetic and environmental factors. It manifests as insufficient insulin secretion from pancreatic β-cells and tissue insulin resistance, leading to hyperglycemia. A sedentary lifestyle, lack of exercise, smoking, and alcohol consumption are the main risk factors for DM. [3, 4] Lipid fibrosis (FL) is a pathological condition of the liver. FL develops when lipid accumulation in hepatocytes exceeds 5% of the liver's wet weight. Unhealthy diet and lack of physical activity are the determining factors for FL. [5, 6] .

[0003] Human glucagon-like peptide-1 (GLP-1) is a 31-amino acid polypeptide that exerts biological effects after binding to its receptor, including suppressing appetite, delaying gastric emptying, inducing weight loss, and regulating inflammation in multiple parts of the body, as well as improving related diseases of the metabolic, cardiovascular, nervous, and digestive systems. [7, 8] .

[0004] Human peptide tyrosine (PYY) is a polypeptide composed of 36 amino acids. 3-36 This is the main form of PYY in the body. After binding to receptors, it exerts biological effects, including inhibiting gastrointestinal motility and pancreatic secretion, altering intestinal permeability, increasing the absorption of water and electrolytes in the colon, and regulating inflammation in multiple parts of the body, improving related diseases of the metabolic, cardiovascular, nervous, and digestive systems. [9, 10] .

[0005] In recent years, the combined use of multiple endogenous peptides or proteins with complementary effects has become a new concept in drug intervention. For example, the combined use of glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter inhibitors has been shown to more effectively treat diabetes. 3-36 Combining with pancreatic amyloid peptides more effectively reduces weight.[11, 12] . Summary of the Invention

[0006] This invention proposes a composition that improves the levels of GLP-1 and / or PYY in obesity, diabetes mellitus, FL, and other conditions. 3-36 It has significant effects on related diseases, including anxiety and depression.

[0007] The present invention also provides the use of the above composition in the preparation of pharmaceuticals.

[0008] The present invention also provides a method for the prevention and / or treatment of a disease.

[0009] The present invention also provides a medicine comprising the above-described composition.

[0010] The present invention also provides the use of the above composition in the preparation of health products, nutritional supplements or as food additives.

[0011] A composition according to a first aspect of the present invention comprises: a GLP-1 peptide analog and PYY 3-36 Peptide analogs; the amino acid sequence of the GLP-1 peptide analog is shown in Formula I; PYY 3-36 The amino acid sequences of the analogues are shown in Formula II; HX 2a X 3a GTF TSD VSS YLE GQA AKX 21a FIA WLX 27a KX 29a RX 31a (Formula I); X 2a Selected from valine, isoleucine, glycine, threonine, leucine, and serine; X 3a Selected from serine and alanine; X 21a Selected from arginine and lysine; X 27a Selected from serine and alanine; X 29a Selected from alanine, serine, threonine, and methionine; X 31a Selected from alanine, serine, threonine, and methionine; IKP EAP GEX 11b ASP EEL X 18b X 19b Y YAX 23b LRH X 27b LN X 30b VT RQR Y (Formula II); X 11b Selected from aspartic acid, glycine, glutamic acid, and asparagine; X 18b Selected from aspartic acid, glutamine, and histidine; X 19b Selected from arginine and lysine; X 23b Selected from serine, threonine, alanine, glycine, and methionine; X 27b Selected from tyrosine, tryptophan, and phenylalanine; X 30b It is selected from leucine, alanine, isoleucine, valine, and methionine.

[0012] According to some embodiments of the present invention, the amino acid sequence of the GLP-1 polypeptide analog is shown in Formula III (SEQ ID NO: 1); HGE GTF TSD VSS YLE GQA AKE FIA WLV KSRA Formula III.

[0013] According to some embodiments of the present invention, the PYY 3-36 The amino acid sequences of the analogues are shown in Formula IV (SEQ ID NO: 2); IKP EAP GED ASP EEL VRY YAG LRH WLN LVT RQRY Formula IV.

[0014] According to some embodiments of the present invention, the GLP-1 polypeptide analog and the PYY 3-36 The mass ratio of analogues is 1:0.25 to 4. For example, it can be 1:0.25, 1:0.5, 1:0.75, 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1:2.25, 1:2.5, 1:2.75, 1:3, 1:3.25, 1:3.5, 1:3.75 or 1:4.

[0015] The composition according to a second aspect of the present invention is used for treating a disease; or for use in the preparation of a medicament for preventing and / or treating a disease.

[0016] According to some embodiments of the present invention, the medicament is used to prevent and / or treat diseases including but not limited to obesity and related complications, diabetes and related complications, impaired glucose tolerance and related diseases, hyperglycemia, hyperinsulinemia, hypertension, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, cardiovascular disease, stroke, diseases related to intestinal permeability disorders such as inflammatory bowel syndrome and / or dyspepsia and / or ulcerative colitis and / or Crohn's disease, stroke and / or hemorrhagic stroke, neuroinflammatory diseases, rheumatoid arthritis, kidney disease, asthma, chronic obstructive pulmonary disease, metabolic syndrome, mood disorders, neurodegenerative diseases, systemic lupus erythematosus, etc.

[0017] According to some embodiments of the present invention, the emotional disorder includes at least one of anxiety disorder and depression.

[0018] According to some embodiments of the present invention, the neurodegenerative disease includes Alzheimer's disease.

[0019] According to some embodiments of the present invention, the drug has at least one of the effects of B1) to B4): B1) Reduce food intake; B2) Reduce weight; B3) Increases insulin sensitivity; B4) Inhibits fat accumulation.

[0020] According to some embodiments of the present invention, the fat accumulation described in B4) includes at least one of subcutaneous fat accumulation, interorgan fat accumulation, and hepatic fat accumulation.

[0021] A method for the prevention and / or treatment of a disease according to a third aspect of the present invention includes administering a therapeutically effective amount of the above-described composition to a subject.

[0022] According to some embodiments of the present invention, the diseases include at least one of the following: obesity and related complications, diabetes and related complications, impaired glucose tolerance and related diseases, hyperglycemia, hyperinsulinemia, hypertension, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, cardiovascular disease, stroke, diseases caused by intestinal permeability disorders such as inflammatory bowel syndrome and / or dyspepsia and / or ulcerative colitis and / or Crohn's disease, stroke and / or hemorrhagic stroke, neuroinflammatory diseases, rheumatoid arthritis, kidney disease, asthma, chronic obstructive pulmonary disease, metabolic syndrome, mood disorders, neurodegenerative diseases, and systemic lupus erythematosus.

[0023] According to some embodiments of the present invention, the emotional disorder includes at least one of anxiety disorder and depression.

[0024] According to some embodiments of the present invention, the neurodegenerative disease includes Alzheimer's disease.

[0025] According to some embodiments of the present invention, the GLP-1 polypeptide analog and the PYY 3-36 The mass ratio of the drug dosage of the analogue is 1:0.25~4.

[0026] A medicament according to a fourth aspect of the present invention comprises the above-described composition, and optionally a pharmaceutically acceptable excipient.

[0027] According to some embodiments of the present invention, the drug dosage form is selected from tablets, capsules, injections, etc. The dosage form is selected to be suitable for the route of administration.

[0028] According to some embodiments of the present invention, the excipient includes at least one of filler, binder, lubricant, solvent, disintegrant, buffer, and preservative.

[0029] The use of the above-described composition according to the fifth aspect of the present invention in the preparation of health products, nutritional supplements or as a food additive.

[0030] The present invention has at least the following beneficial effects: The composition comprises: a GLP-1 peptide analog and PYY 3-36 Peptide analogues. Compared to the native GLP-1 sequence and PYY... 3-36 Natural sequence, GLP-1 peptide analog and PYY 3-36 Compositions of peptide analogs have shown significant effects in improving metabolic diseases such as obesity, diabetes, and fatty liver, as well as mood disorders such as anxiety and depression.

[0031] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0032] Figure 1 The effect of combined use of Formula III (referred to as G1 in the diagram, the same below) and Formula IV (referred to as P1 in the diagram, the same below) on the cytotoxicity of HEK-293t cells in the CCK-8 assay was explained. Figure 2 This illustrates the grouping pattern of the mice in Experiment Example 3; Figure 3 The effects of Formulas III and IV and their combination (referred to as G1P1 in the illustration, the same below) on body weight and food intake in HFD mice are illustrated; a is the rate of change in body weight; b is the rate of change in food intake. Figure 4The effects of Formulas III and IV and their combination on anxiety and depression symptoms in mice were explained; where a and b are the dwell time and number of times in the central region of the open field experiment, c is the dwell time in the open arm region of the cross maze experiment, and d is the immobility time in the tail suspension experiment. Figure 5 This demonstrates the effects of Formulas III and IV, and their combination, on blood glucose levels in HFD mice during the GTT experiment; Figure 6 This demonstrates the effects of Formulas III and IV, and their combination, on blood glucose levels in HFD mice during the ITT experiment; Figure 7 This study illustrates the effects of Formulas III and IV and their combined use on fatty liver in the Oil Red O staining experiment, where a represents Oil Red O staining of the liver and b represents the quantification of Oil Red O staining of the liver. Figure 8 This describes the effects of Formulas III and IV, and their combined use, on muscle in HE staining experiments; where a represents HE staining of muscle, and b represents HE quantification of muscle. Figure 9 This describes the effects of Formulas III and IV, and their combination, on body weight in investigational clinical trials; where a represents body weight change and b represents the rate of body weight change. Detailed Implementation

[0033] The following description illustrates exemplary embodiments of the present invention. It should be understood that such description is not intended to limit the scope of the invention, but rather is provided as a description of exemplary embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are all within the scope of protection of the present invention.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0036] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0037] Unless otherwise specified, the quantities of different components and reaction conditions used herein are to be interpreted as "approximate" or "about". Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0038] As used herein, the term "GLP-1 analogue" refers to an analogue of GLP-1 or a variant thereof, used to refer to polypeptides of formula I or III. The "GLP-1 analogue" possesses GLP-1 activity. GLP-1 activity refers to the ability to bind to the GLP-1 receptor and trigger signal transduction pathways to produce insulin-stimulating effects or other physiological effects.

[0039] The term "PYY" used in this article 3-36 "Analog" refers to an analogue of PYY or a variant thereof, used to refer to polypeptides of formula II or IV. The "PYY" 3-36 "Analogs" have PYY 3-36 Activity. Possesses PYY 3-36 Activity refers to the interaction with PYY 3-36 The ability of receptors to bind and trigger signal transduction pathways to produce insulin-promoting effects or other physiological effects.

[0040] The GLP-1 analogue and PYY of the present invention 3-36 Analogs can be prepared using various peptide synthesis techniques well-known in the art. [13-15] The instruments, equipment, and reagents required for the synthesis can all be obtained commercially through conventional means. The GLP-1 analogue and PYY of this invention... 3-36 Analogs can be synthesized stepwise from the C-terminus of the peptide on an insoluble polymer support (also referred to as a "resin"). The synthesis begins by attaching the C-terminal amino acid of the peptide to the resin via the formation of an amide or ester bond. This results in the final release of the peptide as either a C-terminal amide or a carboxylic acid, respectively. Alternatively, if the C-terminal amino acid is present, the C-terminal residue as described herein can be linked to a 2-methoxy-4-alkoxybenzyl alcohol resin, and after the peptide sequence assembly is complete, the resulting peptide is released with a solution of lithium borohydride in tetrahydrofuran.

[16] .

[0041] Those skilled in the art know that amino acids exist in two isomers, D and / or L, and the present invention includes the use of one isomer and / or a mixture of the other isomers of amino acids in the synthesis of the peptides described herein.

[0042] The GLP-1 analogue and PYY of the present invention 3-36Analogs can also be produced via genetic recombination, i.e., by culturing host cells containing the deoxyribonucleic acid sequence encoding the analog and capable of expressing the peptide in a suitable nutrient medium under conditions that allow for peptide expression. Non-limiting examples of suitable host cells for expressing these peptides include: *Escherichia coli* (…). Escherichia coli ), brewer's yeast ( Saccharomyce scerevisiae And mammalian juvenile hamster kidney cell lines or Chinese hamster ovary cell lines. In some embodiments, this completely recombinant fermentation step in the production process is desirable, for example, for economic reasons. It will contain GLP-1 analogs or PYY 3-36 The fusion protein inclusion bodies of the analog backbone undergo denaturation and renaturation to obtain fusion proteins with the correct conformation. After a series of treatments including enzyme digestion, sedimentation, and centrifugation, high-content GLP-1 analogs or PYY analogs are obtained. 3-36 The analog backbone was further purified by ion exchange chromatography to obtain a high-purity GLP-1 analog or PYY. 3-36 Analogous main chain.

[0043] The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. Excipients can be pharmaceutically acceptable inert, inactive, and / or non-pharmacologically active substances. Formulation of active pharmaceutical ingredients with different excipients is known in the art. Non-limiting examples of excipient functions include, but are not limited to, fillers, solvents, etc. Those skilled in the art can readily select excipients with suitable functions based on the formulation; suitable carriers can be found in [reference needed].

[17] The term "filler" includes, but is not limited to, lactose, sucrose, etc.; "solvent" includes, but is not limited to, ethanol, water, etc.

[0044] The compositions of the present invention can be administered pharmaceutically in a variety of dosage forms (each dosage form includes immediate-release, sustained-release, or controlled-release dosage forms), including but not limited to tablets, capsules, and injections. Those skilled in the art can select a suitable dosage form based on the route of administration. The routes of administration include, but are not limited to, oral, transdermal, subcutaneous, intravenous, and intramuscular administration. The dosage forms are formulated according to the routes of administration based on acceptable pharmaceutical practices.

[18] All dosage forms used are familiar to those skilled in the pharmaceutical field, and pharmaceutically acceptable excipients are selected and mixed together in accordance with the chosen route of administration and standard pharmaceutical procedures before administration.

[0045] The compositions of the present invention can also be administered in combination with soluble polymers, such as conjugated with targeted drug carriers, with biodegradable polymers for controlled drug release, and with crosslinked or amphiphilic block copolymers of hydrogels.

[0046] The term "treatment of disease" refers to the treatment and care of patients who already have a disease, condition, or disorder. The goal of treatment is to combat the disease, condition, or disorder. Treatment includes the administration of active compounds to eliminate or control the disease, condition, or disorder, and to alleviate symptoms or complications associated with the disease, condition, or disorder.

[0047] The dosing regimen of the formulations of this invention will be modified based on known factors such as the pharmacokinetic properties of the specific drug and its administration mode and route, the recipient's species, health status, medical status, age, sex, and weight, the nature and severity of the symptoms, the type and frequency of treatment, the patient's renal and hepatic function, and the desired effect. A physician or veterinarian can prescribe the drug based on the amount required to effectively prevent, counteract, or inhibit the progression of the disease state.

[0048] Administration of the therapeutic agents of the present invention includes, but is not limited to, administration at a therapeutically effective amount of the agent described herein. The term "therapeutically effective amount" refers to the amount of a therapeutic agent used to treat and / or prevent conditions that can be treated by administration of the composition of the present invention. This dose is sufficient to exhibit a therapeutic, preventative, or ameliorative effect. Such effects include the treatment and / or prevention of the conditions listed herein. The precise effective amount used on a subject will depend on the subject's weight and health status, the nature and severity of the condition to be treated, the advice of the treating physician, and the chosen method or combination of treatments for administration. Therefore, specifying an exact effective amount beforehand is not helpful.

[0049] The term "unit dose" in this invention refers to the fact that the compositions of this invention are in unit dose form, which may be unit dose forms commonly used in pharmaceutical formulations. Unit dose refers to the amount of active pharmaceutical ingredient contained in each tablet, capsule, or injection.

[0050] In accordance with general guidelines, the active ingredient in the composition will typically be present in an amount of about 0.5-95% based on the total weight of the composition. The compositions of the present invention can be administered as a single daily dose, or a total daily dose divided into two, three, or four daily doses. The compositions of the present invention can also be administered as long-acting formulations, which will allow the active ingredient to be slowly released over several days / weeks / months as needed.

[0051] abbreviations

[0052] References [1]Lister N B, Baur L A, Felix J F, et al. Child and adolescentobesity [J]. Nat Rev Dis Primers, 2023, 9(1): 24. [2]Safaei M, Sundararajan E A, Driss M, et al. A systematicliterature review on obesity: Understanding the causes&consequences ofobesity and reviewing various machine learning approaches used to predictobesity [J]. Computers in Biology and Medicine, 2021, 136: 104754. [3]Tomic D, Shaw J E, Magliano D J. The burden and risks of emergingcomplications of diabetes mellitus [J]. Nat Rev Endocrinol, 2022, 18(9): 525-39. [4]Antar S A, Ashour N A, Sharaky M, et al. Diabetes mellitus:Classification, mediators, and complications; A gate to identify potentialtargets for the development of new effective treatments [J]. BiomedPharmacother, 2023, 168: 115734. [5]Loomba R, Friedman S L, Shulman G I. Mechanisms and diseaseconsequences of nonalcoholic fatty liver disease [J]. Cell, 2021, 184(10):2537-64. [6]Powell E E, Wong V W, Rinella M. Non-alcoholic fatty liver disease[J]. Lancet, 2021, 397(10290): 2212-24. [7]Drucker D J. GLP-1 physiology informs the pharmacotherapy ofobesity [J]. Mol Metab, 2022, 57: 101351. [8]Drucker D J. The benefits of GLP-1 drugs beyond obesity [J].Science, 2024, 385(6706): 258-60. [9]Chen W, Binbin G, Lidan S, et al. Evolution of peptide YY analogsfor the management of type 2 diabetes and obesity [J]. Bioorg Chem, 2023,140: 106808.

[10] Samms R J, Cosgrove R, Snider B M, et al. GIPR Agonism InhibitsPYY-Induced Nausea-Like Behavior [J]. Diabetes, 2022, 71(7): 1410-23.

[11] Boland B B, Laker R C, O'Brien S, et al. Peptide-YY(3-36) / glucagon-like peptide-1 combination treatment of obese diabetic mice improvesinsulin sensitivity associated with recovered pancreatic β-cell function andsynergistic activation of discrete hypothalamic and brainstem neuronalcircuitries [J]. Mol Metab, 2022, 55: 101392.

[12] Dischinger U, Hasinger J, Königsrainer M, et al. Toward a MedicalGastric Bypass: Chronic Feeding Studies With Liraglutide + PYY(3-36)Combination Therapy in Diet-Induced Obese Rats [J]. Front Endocrinol(Lausanne), 2020, 11: 598843.

[13] Gross E. The Peptides Analysis, Synthesis, Biology: ModernTechniques of Conformational Structural, and Configurational Analysis [M].Elsevier, 2012.

[14] Schröder E, Lübke K. The Peptides: Methods of peptide synthesis[M]. Elsevier, 2014.

[15] Chan W, White P. Fmoc solid phase peptide synthesis: a practicalapproach [M]. OUP Oxford, 1999.

[16] Collins J M, Singh S K, White T A, et al. Total wash eliminationfor solid phase peptide synthesis [J]. Nat Commun, 2023, 14(1): 8168.

[17] Paul J. S. Handbook of Pharmaceutical Excipients [M].Pharmaceutical Press, 2020.

[18] Goodman L S. Goodman and Gilman's the pharmacological basis oftherapeutics [M]. McGraw-Hill New York, 2022. Example 1 This example provides a composition comprising a GLP-1 peptide analog and PYY 3-36 Composition of polypeptide analogs; The amino acid sequence of the GLP-1 polypeptide analog is shown in Formula I; HX 2a X 3a GTF TSD VSS YLE GQA AKX 21a FIA WLX 27a KX 29a RX 31a (Formula I); X 2a Selected from valine, isoleucine, glycine, threonine, leucine, and serine; X 3a Selected from serine and alanine; X 21a Selected from arginine and lysine; X 27a Selected from serine and alanine; X 29a Selected from alanine, serine, threonine, and methionine; X 31a Selected from alanine, serine, threonine, and methionine; PYY 3-36 The amino acid sequences of the analogues are shown in Formula II. IKP EAP GEX 11b ASP EEL X 18b X 19b Y YAX 23b LRH X 27b LN X 30b VT RQR Y (Formula II); X 11b Selected from aspartic acid, glycine, glutamic acid, and asparagine; X 18b Selected from aspartic acid, glutamine, and histidine; X 19b Selected from arginine and lysine; X 23b Selected from serine, threonine, alanine, glycine, and methionine; X 27b Selected from tyrosine, tryptophan, and phenylalanine; X 30b It is selected from leucine, alanine, isoleucine, valine, and methionine.

[0053] Without altering the secondary structure of Formula I or Formula II, specific site substitutions of amino acids are achieved based on the principle of conservative substitution of amino acids.

[0054] Example 2 This example provides a composition comprising a GLP-1 peptide analog and PYY 3-36 Composition of polypeptide analogs; The amino acid sequence of the GLP-1 peptide analog is shown in Formula III (SEQ ID NO: 1); HGE GTF TSD VSS YLE GQA AKE FIA WLV KSRA Formula III.

[0055] PYY 3-36 The amino acid sequence of the analogue is shown in Formula IV (SEQ ID NO: 2). IKP EAP GED ASP EEL VRY YAG LRH WLN LVT RQRY Formula IV.

[0056] Example 3: Cytotoxicity of combined use of Formula III and Formula IV drugs High-glucose DMEM complete medium (Wuhan Saive Biotechnology, hereinafter the same) was used to prepare mixed molar concentration gradient solutions of Formula III and Formula IV, with molar concentration gradients of 2 μmol / mL (meaning both Formula III and Formula IV concentrations are 2 μmol / mL, hereinafter the same), 1 μmol / mL, 0.5 μmol / mL, 0.25 μmol / mL, and 0.125 μmol / mL, respectively. HEK-293t cells (5000 cells / 100 μL / well) were added to 96-well plates (37±0.5℃, 5±0.1% CO2) and cultured for 24 h. Then, 100 μL of mixed molar concentration gradient solutions of Formula III and Formula IV were added to each well. The absorbance at 450 nm was measured using a CCK-8 assay kit (Wuhan Saive Biotechnology), and cell viability was calculated. IC50 was plotted using GraphPadPrism. 50 curve.

[0057] The results are as follows Figure 1 As shown.

[0058] When Equations III and IV are used in equal proportions, their IC 50 It is 0.57 μmol / L.

[0059] Example 4: Effect of combined administration of Formula III and Formula IV on mouse body weight Fifty SPF-grade male C57BL / 6 mice (Beijing Spefol Biotechnology Co., Ltd., hereinafter the same) were housed in an environment with a temperature of 22–26℃ and a relative humidity of 40%–70%, with ventilation of 8–12 times / h. They were randomly divided into 5 groups (10 mice / group, e.g.) Figure 2 The groups are: control group (referred to as Control in the figure, the same below), model group (referred to as Model in the figure, the same below), group III (referred to as G1 group in the text, and G1 in the figure, the same below), group IV (referred to as P1 group in the text, and P1 in the figure, the same below), and group using a combination of formulas III and IV (referred to as G1P1 group in the text, and G1P1 in the figure, the same below).

[0060] From day 1 to day 7, the model group, G1 group, P1 group, and G1P1 group were given a 60%–80% high-fat diet (HFD) for dietary adaptation induction. From day 8 to day 55, they were given a 100% high-fat diet. From day 15 to day 55, the model group was injected subcutaneously with 0.9% normal saline (NS), the G1 group was injected with 60 μmol / kg of formula III, the P1 group was injected with 80 μmol / kg of formula IV, and the G1P1 group was injected with both 60 μmol / kg of formula III and 80 μmol / kg of formula IV, for a total of 40 days. C57BL / 6 mice were administered the drugs on an empty stomach, and daily body weight changes and total food intake 48 hours after each administration were measured.

[0061] Test results as follows Figure 3 As shown.

[0062] Starting on day 15, significant differences in body weight were observed among the five groups of mice. Compared to the control group, the model group showed a significant increase in body weight of 40%-70%. Compared to the model group, the G1, P1, and G1P1 groups showed weight reductions of approximately 5%-45%, 5%-30%, and 5%-55%, respectively. When fed a high-fat diet, the G1P1 group showed weight reductions of approximately 5%-20% and 5%-45% compared to the G1 and P1 groups, respectively. Compared with the control group, the food intake of the model group increased significantly by 30%-75%. Compared with the model group, the food intake of the G1 group, P1 group and G1P1 group decreased by about 5%-50%, 5%-45% and 5%-60%, respectively; compared with the G1 group and P1 group, the food intake of the G1P1 group decreased by about 5%-60% and 5%-45%, respectively.

[0063] Example 5: Effects of G1P1 combination therapy on anxiety and depression Behavioral experiments were conducted on each group of mice in Example 4.

[0064] Open Field Test (OFT): Mice are placed in a test chamber and their activity is monitored during the test period. Elevated Plus Maze Test (EPM): Mice are placed in the center of an elevated plus maze and their activity is observed during the test period. Tail Suspension Test (TST): Mice are suspended by their tails on a cage frame and their struggling, swinging, and stillness patterns are observed.

[0065] The results are as follows Figure 4 As shown.

[0066] In the OFT and EPM experiments, compared with the model group, the control group, G1 group, P1 group and G1P1 group mice all showed significantly increased movement time and frequency in the central region and significantly increased movement time in the open arm region; compared with the control group, G1 group and P1 group, the G1P1 group showed increased movement time and frequency in the central region and increased movement time in the open arm region.

[0067] In the TST experiment, the immobility time of mice in the model group was increased compared with that of mice in the control group, G1 group, P1 group and G1P1 group; the immobility time of mice in the G1P1 group was decreased compared with that of mice in the control group, G1 group and P1 group.

[0068] The results above show that obesity exacerbates anxiety and depression symptoms in mice. Administration of G1P1 in combination with other drugs significantly improved or even prevented anxiety and depression symptoms in mice compared to administration of G1 or P1 alone. Combined results from the comparison between the G1P1 group and the control group indicate that G1P1 in combination with other drugs effectively prevents anxiety and depression in normal mice.

[0069] Example 6: Effect of G1P1 combination therapy on glucose tolerance The steps for the Glucose Tolerance Test (GTT) are as follows: This experiment was conducted on mice from the group in Example 5 after they recovered. The mice were fasted for one day before the experiment, but had normal water intake during the period. They were administered a 20% glucose aqueous solution by gavage at a volume of 10 μL / g (based on body weight). Blood glucose levels were measured using a glucometer at 15 min, 30 min, 60 min, 90 min, and 120 min.

[0070] The results of the blood glucose meter test are as follows Figure 5 As shown.

[0071] The blood glucose levels in the model group increased by approximately 5%-95%, 5%-60%, 5%-55%, and 5%-80% compared to the control group, G1 group, P1 group, and G1P1 group, respectively, within 0 min-120 min. The blood glucose levels in the G1P1 group decreased by approximately 5%-35% compared to the G1 group and by approximately 10%-45% compared to the P1 group within the same period, indicating that the combination therapy with G1P1 is more effective than monotherapy in lowering blood glucose.

[0072] Example 7: Effect of G1P1 combination therapy on insulin glucose tolerance The steps of the Insulin Tolerance Test (ITT) are as follows: The mice were given a 3-day recovery period after completing Example 6 before this experiment. The mice were fasted for one day before the experiment, but had normal water intake during the period. Each mouse was injected intraperitoneally with 0.75 IU / kg of insulin solution, and blood glucose levels were measured using a blood glucose meter and blood glucose test strips at 15 min, 30 min, 60 min, 90 min, and 120 min.

[0073] The results of the blood glucose meter test are as follows Figure 6 As shown.

[0074] Within 0-120 minutes, blood glucose levels decreased as follows: control group approximately 5%-85%, model group approximately 5%-30%, G1 group approximately 5%-55%, P1 group approximately 5%-65%, and G1P1 group approximately 10%-75%. Compared with the model group, insulin tolerance improved in the G1, P1, and G1P1 groups 2 hours after intraperitoneal insulin injection. Blood glucose levels in the G1P1 group decreased by approximately 5%-20% and 5%-10% compared to the G1 and P1 groups, respectively, indicating that G1P1 combination therapy is more effective than single-drug therapy in improving insulin tolerance.

[0075] Example 8: Effects of G1P1 combination therapy on fat accumulation and fatty liver in the body After completing Example 7, mice were directly anesthetized, fixed supine on a mouse board, and their skin was dissected to expose subcutaneous fat. The accumulation of subcutaneous fat was recorded. Then, the organs were exposed to observe fat accumulation between organs. Finally, the leg muscles and liver tissue of the mice were collected and stored at -80°C.

[0076] In the model group, subcutaneous and interorgan fat accumulation was approximately 35%-65% higher than in the control group. In the G1P1 group, subcutaneous and interorgan fat accumulation was 10%-20% higher than in the control group, approximately 5%-30% lower than in the model group, and approximately 5%-10% lower than in the G1 and P1 groups. This indicates that the G1P1 group is significantly effective in reducing subcutaneous and interorgan fat accumulation, and that the combination of G1P1 and other medications is more effective than single-drug therapy in controlling fat accumulation in the body.

[0077] Example 9: Effects of G1P1 combination therapy on fatty liver The mouse liver tissue collected in Example 8 was cryosectioned. The liver tissue sections were fixed with formaldehyde-calcium, washed, and then immersed in 60% isopropanol. Saturated Oil Red O stock solution (Beijing Solarbio Science & Technology) was diluted at a ratio of 3:2 (Oil Red O: distilled water), stained, mounted with glycerol gelatin, and examined under a microscope.

[0078] The results are as follows Figure 7 As shown.

[0079] In Oil Red O staining of mouse liver tissue, compared with the control group, the content of fat droplets in the model group was significantly increased by approximately 70%-99%. Compared with the model group, the content of fat droplets in the G1 group, P1 group, and G1P1 group was significantly reduced by approximately 40%-65%, 40%-55%, and 50%-85%, respectively. The content of fat droplets in the G1P1 group was reduced by approximately 10%-45% and 10%-60% compared with the G1 group and P1 group, respectively. This shows that the combination of P1G1 and P1 is significantly effective in reducing hepatic fat accumulation and is more effective than single-drug therapy in treating fatty liver.

[0080] Example 10: Effects of G1P1 combination therapy on muscle The mouse muscle tissue collected in Example 8 was cryosectioned. It was stained with hematoxylin and eosin (Beijing Solarbio Science & Technology), mounted with glycerol gelatin, and examined under a microscope.

[0081] The results are as follows Figure 8 As shown.

[0082] Compared with the control group, the muscle surface area of ​​mice in the model group, G1 group, P1 group and G1P1 group increased by 5%-20%, 5%-15%, 5%-10% and 5%-20%, respectively. Compared with the model group, there was no significant difference in muscle area among mice in the G1 group, P1 group and G1P1 group.

[0083] Example 11: Investigational human trial of G1P1 in combination with other drugs With informed consent, qualified volunteers (with no adverse reactions before medication) were recruited to compare the efficacy of P1, G1 alone, and G1P1 combination therapy in weight control in overweight or obese individuals.

[0084] Blinding: Single-blind. Eight volunteers were randomly assigned to four groups, each receiving either a capsule containing the corresponding drug or a placebo (manufactured by Junyang Biopharmaceutical (Shenzhen) Co., Ltd.), once daily for 30 days. Volunteers were weighed before administration and took the capsule with 50-100 mL of drinking water 1.5-2 hours after breakfast daily, between 9:45 AM and 10:15 AM. Volunteers were advised against drinking large amounts of water or eating for 30 minutes after administration; thereafter, they could eat and drink normally.

[0085] The criteria for selecting volunteers are shown in Table 1.

[0086] Table 1. Volunteer Selection Criteria

[0087] The experimental groups are shown in Table 2.

[0088] Table 2 Human Trial Grouping

[0089] The evaluation criteria are shown in Table 3.

[0090] Table 3 Evaluation Criteria for Human Trials

[0091] The results are shown in Table 4 and Figure 9 As shown.

[0092] Table 4. Statistical table of body weight data during human trials

[0093] From a gender perspective, regarding efficacy, during days 0-30, men in the G1, P1, and G1P1 treatment groups experienced weight losses of approximately 0.1 kg-1.2 kg, 0.1 kg-1.0 kg, and 0.1 kg-3.5 kg, respectively, representing percentage reductions of 0.1%-1.3%, 0.1%-1.4%, and 0.1%-3%. Women in the same groups experienced weight losses of approximately 0.1 kg-1.6 kg, 0.1 kg-2.3 kg, and 0.1 kg-3.0 kg, respectively, representing percentage reductions of 0.1%-2.2%, 0.2%-3.6%, and 0.5%-3.4%. Considering similar BMIs, different administration methods (G1P1 combination therapy and G1 or P1 monotherapy) resulted in better weight control in women than in men. Furthermore, G1P1 combination therapy was superior to G1 or P1 monotherapy.

[0094] From a BMI perspective, regarding effectiveness, during days 0-30, when BMI = 24-28 kg / m² 2 At that time, the body weight of the G1 and P1 treatment groups decreased by approximately 0.1 kg-1.4 kg and 0.1 kg-1.7 kg, respectively, with weight loss percentages of 0.1%-1.7% and 0.5%-2.5%. When BMI > 28 kg / m² 2During treatment, the weight loss in the G1P1 group ranged from approximately 0.3 kg to 3.5 kg, with a weight loss percentage of 0.2% to 3.4%. Regarding weight control, the higher the BMI, the more significant the weight control effect of G1P1 in combination with other drugs and G1 or P1 alone. The combination of G1P1 with other drugs was superior to the use of G1 or P1 alone.

[0095] In summary, the combination of G1, P1, and G1P1 demonstrated efficacy, resulting in at least a 1% weight loss over a 30-day treatment period, meeting the expected outcome. Higher BMI correlated with better weight control. At similar BMI levels, women showed better weight control than men. Furthermore, the combination of G1P1 and G1P1 was more effective than either G1 or P1 alone.

[0096] Example 12: Preparation of G1P1 combination drug formulation A) Tablets: Tablets can be prepared according to conventional tablet preparation procedures to obtain tablets of the stated formulation specifications. For example, the formulation may contain 14 g of Formula III, 14 g of Formula IV, 0.02 g of colloidal silica, 0.5 g of magnesium stearate, 27.5 g of microcrystalline cellulose, 1.1 g of starch, and 9.88 g of lactose. Appropriate coatings can be used to improve palatability or delay absorption, and tablets can be prepared in 1000-tablet packs.

[0097] B) Capsules: Capsules can be prepared according to conventional capsule preparation procedures to obtain the specified formulation. For example, 14 g of Formula III, 14 g of Formula IV, 15 g of lactose, 5 g of microcrystalline cellulose, 0.6 g of magnesium stearate, and an appropriate amount of absorption enhancer are filled into standard hard gelatin capsules to prepare 1000 capsules.

[0098] C) Injectable: Injectable preparations can be made using conventional procedures, such as preparing an injectable formulation with the following composition according to the methods described below.

[0099] Dissolve 5 g of Formula III and 5 g of Formula IV in a portion of water for injection. Add 40 g of sulfobutylcyclodextrin to the drug solution and stir for approximately 8 minutes. Adjust the pH to between 6 and 8 by adding sodium hydroxide or hydrogen chloride. Add water for injection and sodium chloride aqueous solution to make the final volume 1 L and ensure isotonicity. Then dispense into suitable ampoules to prepare 1000 injection vials. Before adjusting the pH, other inactive components may be added as needed, such as solvents and cosolvents, solubilizers, emulsifiers or thickeners, chelating agents, antioxidants and reducing agents, antimicrobial preservatives, buffers, swelling agents, protective agents, tension modifiers, and special additives.

[0100] Injectable formulations must be sterile, pyrogen-free, and free of particulate matter in solution. They can be prepared by stirring in a pharmaceutically acceptable buffer, with or without solubilizers or other excipients, and the solution should be isotonicized and sterilized with pharmaceutical-grade sodium chloride before use.

[0101] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A composition, characterized in that, include: GLP-1 peptide analogues and PYY 3-36 peptide analogs; The amino acid sequence of the GLP-1 peptide analog is shown in Formula I; PYY 3-36 The amino acid sequences of the analogues are shown in Formula II; HX 2a X 3a GTF TSD VSS YLE GQA AKX 21a FIA WLX 27a KX 29a R X 31a (Formula I); X 2a Selected from valine, isoleucine, glycine, threonine, leucine, and serine; X 3a Selected from serine and alanine; X 21a Selected from arginine and lysine; X 27a Selected from serine and alanine; X 29a Selected from alanine, serine, threonine, and methionine; X 31a Selected from alanine, serine, threonine, and methionine; IKP EAP GEX 11b ASP EEL X 18b X 19b Y YAX 23b LRH X 27b LN X 30b VT RQR Y (Formula II); X 11b Selected from aspartic acid, glycine, glutamic acid, and asparagine; X 18b Selected from aspartic acid, glutamine, and histidine; X 19b Selected from arginine and lysine; X 23b Selected from serine, threonine, alanine, glycine, and methionine; X 27b Selected from tyrosine, tryptophan, and phenylalanine; X 30b It is selected from leucine, alanine, isoleucine, valine, and methionine.

2. The composition according to claim 1, characterized in that, The amino acid sequence of the GLP-1 polypeptide analog is shown in Formula III; HGE GTF TSD VSS YLE GQA AKE FIA WLV KSRA Formula III; And / or, the PYY 3-36 The amino acid sequences of the analogues are shown in Formula IV. IKP EAP GED ASP EEL VRY YAG LRH WLN LVT RQRY Formula IV.

3. The composition according to claim 1, characterized in that, The GLP-1 polypeptide analog and the PYY 3-36 The mass ratio of the analogues is 1:0.25~4.

4. The use of the composition according to any one of claims 1 to 3 in A1) or A2); A1) To prepare medicines for the prevention and / or treatment of diseases; A2) Prevention and / or treatment of diseases.

5. The application according to claim 4, characterized in that, The diseases mentioned include at least one of the following: obesity and related complications, diabetes and related complications, impaired glucose tolerance and related diseases, hyperglycemia, hyperinsulinemia, hypertension, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, cardiovascular disease, stroke, diseases caused by intestinal permeability disorders such as inflammatory bowel syndrome and / or dyspepsia and / or ulcerative colitis and / or Crohn's disease, stroke and / or hemorrhagic stroke, neuroinflammatory diseases, rheumatoid arthritis, kidney disease, asthma, chronic obstructive pulmonary disease, metabolic syndrome, mood disorders, neurodegenerative diseases, and systemic lupus erythematosus. Preferably, the emotional disorder includes at least one of anxiety disorder and depression; Preferably, the neurodegenerative disease includes Alzheimer's disease.

6. The application according to claim 4, characterized in that, The drug has at least one of the effects of B1) to B4): B1) Reduce food intake; B2) Reduce weight; B3) Increases insulin sensitivity; B4) Inhibits fat accumulation; Preferably, the fat accumulation described in B4) includes at least one of subcutaneous fat accumulation, interorgan fat accumulation, and hepatic fat accumulation.

7. A method for the prevention and / or treatment of a disease, characterized in that, The invention includes administering a therapeutically effective amount of the composition according to any one of claims 1 to 3 to a subject; preferably, the disease includes at least one of obesity and related complications, diabetes and related complications, impaired glucose tolerance and related diseases, hyperglycemia, hyperinsulinemia, hypertension, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, cardiovascular disease, stroke, related diseases caused by intestinal permeability disorders such as inflammatory bowel syndrome and / or dyspepsia and / or ulcerative colitis and / or Crohn's disease, stroke and / or hemorrhagic stroke, neuroinflammatory diseases, rheumatoid arthritis, nephropathy, asthma, chronic obstructive pulmonary disease, metabolic syndrome, mood disorders, neurodegenerative diseases, and systemic lupus erythematosus; Preferably, the emotional disorder includes at least one of anxiety disorder and depression; Preferably, the neurodegenerative disease includes Alzheimer's disease.

8. A drug, characterized in that, It includes the composition according to any one of claims 1 to 3, and optionally a pharmaceutically acceptable excipient.

9. The medicament according to claim 8, characterized in that, The drug dosage form is selected from tablets, capsules, and injections.

10. The use of the composition according to any one of claims 1 to 3 in the preparation of health products, nutritional supplements or as a food additive.

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