PYY analogue and application thereof in treating diseases

Abstract

The invention relates to the field of medicines, and discloses a novel human PYY analogue, and compared with a PYY3-36 natural sequence, the novel human PYY analogue has a remarkable effect on improving metabolic diseases such as obesity, diabetes and fatty liver and emotional diseases such as anxiety disorder and depression.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a PYY analogue and its application in disease treatment. Background Technology

[0002] Peptide tyrosine (PYY) is a 36-amino acid peptide that is mainly released by L cells in the distal gastrointestinal tract, ileum, and colon. [1] PYY belongs to the neuropeptide Y (NPY) family and can bind extensively to the neuropeptide Y receptor (NPYR). PYY contains PYY... 1-36 and PYY 3-36 Two endogenous forms, PYY 3-36 It is generated by the cleavage of dipeptidyl peptidase IV (DPP-IV) and has the strongest binding affinity to NPY2R. [1] Due to NPYR mediation [2] PYY 3-36 It plays a role in suppressing appetite and promoting insulin secretion, inhibiting gastrointestinal motility and pancreatic secretion, regulating intestinal peristalsis and inflammation in multiple sites, and improving related diseases of the metabolic, cardiovascular, nervous and digestive systems. [3,4] Given the widespread expression of the PYY receptor NPYR in vivo, PYY 3-36 It has received considerable attention in research on diseases such as obesity, diabetes, musculoskeletal disorders, and cancer.

[0003] Obesity and its associated diseases have become a major global health problem. [5] For example, gallbladder cancer, kidney cancer, thyroid cancer, leukemia, liver cancer, and ovarian cancer are all associated with obesity. [6] Obesity can lead to chronic diseases such as diabetes mellitus (DM), fatty liver (FL), cardiovascular disease, and mental disorders. [7-9] The World Health Organization (WHO) defines obesity as "an abnormal or excessive accumulation of fat that may impair health."

[10] Excessive alcohol consumption, lack of exercise, and overeating can lead to excessive fat accumulation and obesity.

[11] Diabetes mellitus (DM) is a chronic metabolic disease characterized by insufficient insulin secretion from pancreatic β-cells and tissue insulin resistance, leading to hyperglycemia. Obesity, sedentary lifestyle, high-calorie diets, and an aging population are the main driving factors for DM. Liver fibrosis (FL) is a disease characterized by pathological changes in the liver; FL forms when liver cells contain more than 5% triglycerides.

[12] Excessive alcohol consumption and high-calorie food intake are the main causes of chronic liver disease.

[13] .

[0004] PYY 3-36 It is easily degraded in the body and is not suitable for therapeutic use. Currently, long-acting PYY... 3-36 Analogs (NN9748 and NNC0165-1875) are in Phase I clinical trials for the treatment of obesity, and the combination of NNC0165-1875 and smegglutide is in Phase II clinical trials. Summary of the Invention

[0005] This invention provides a new PYY 3-36 Analogs, derived from human-based PYY 3-36 Mutations at specific sites in sequence fragments, and PYY 3-36 Compared to its natural form, it works more effectively in the body, including but not limited to improving conditions such as obesity, diabetes, and fatty liver. It can also significantly improve symptoms of mood disorders such as anxiety and depression.

[0006] To achieve the above objectives, the first aspect of the present invention provides a PYY 3-36 Analog, the PYY 3-36 Analogs include the amino acid sequence shown in Formula I:

[0007] IKP EAP GEX 11 ASP EEL X 18 X 19 Y YAX 23 LRH X 27 LN X 30 VT RQRY

[0008] Formula I

[0009] Without altering the secondary structure of Formula I, and based on the principle of conservative amino acid substitution, specific site substitutions of amino acids are achieved, wherein...

[0010] X 11 Selected from aspartic acid, glycine, glutamic acid, and asparagine;

[0011] X 18 Selected from aspartic acid, glutamine, and histidine;

[0012] X 19 Selected from arginine and lysine;

[0013] X 23 Selected from serine, threonine, alanine, glycine, and methionine;

[0014] X 27Selected from tyrosine, tryptophan, and phenylalanine;

[0015] X 30 It is selected from leucine, alanine, isoleucine, valine, and methionine.

[0016] In some implementations, PYY 3-36 Analogs include the polypeptide sequence shown in Formula II:

[0017] IKP EAP GED ASP EEL VRY YAG LRH WLN LVT RQRY(SEQ ID NO:1)

[0018] Formula II.

[0019] A second aspect of the invention provides a composition comprising PYY as described in Formula I or Formula II. 3-36 Analogs and pharmaceutically acceptable excipients.

[0020] The third aspect of the present invention provides the PYY 3-36 The use of the analogue or the composition for treating diseases; or the PYY 3-36 Use of the analogue or the composition in the preparation of a medicament for treating a disease; or a treatment method for a disease comprising administering an effective amount of the PYY to a subject. 3-36 Analogs or the composition thereof.

[0021] In some embodiments, the present invention provides the PYY 3-36 Use of the analogue or the composition for the treatment of NPYR-mediated diseases; or the PYY 3-36 Use of the analogue or the composition in the preparation of a medicament for treating NPYR-mediated diseases; or a treatment method for NPYR-mediated diseases comprising administering an effective amount of the PYY to a subject. 3-36 Analogs or the composition thereof.

[0022] In some embodiments, the NPYR-mediated diseases include obesity and related complications, diabetes and related complications, glucose intolerance 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, anxiety disorders, depression and other mood disorders, and neurodegenerative diseases such as Alzheimer's disease.

[0023] This invention provides the PYY 3-36 The use of analogues or the composition in the preparation of medicaments that reduce food intake and increase insulin sensitivity.

[0024] This invention provides the PYY 3-36 The use of analogues or the composition in the preparation of drugs for inhibiting fat accumulation.

[0025] In some embodiments, the fat accumulation includes subcutaneous fat accumulation, interorgan fat accumulation, and hepatic fat accumulation.

[0026] This invention provides the PYY 3-36 The use of analogues or the compositions thereof in the preparation of health products, nutritional supplements and as food additives.

[0027] 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.

[0028] Beneficial effects:

[0029] This invention discloses a novel human PYY 3-36 Analogs, similar to PYY 3-36 Compared to the natural sequence, it is significantly effective in improving diseases such as obesity, diabetes, and fatty liver, as well as mood disorders such as anxiety and depression. Attached Figure Description

[0030] Figure 1 This study illustrates the effect of peptide II (referred to as P1 in the illustration, the same below) on the cytotoxicity of HEK-293t cells in a CCK-8 assay.

[0031] Figure 2 This study explains the effect of the formula II peptide on cAMP content in the detection assay using the cyclized AMP kit.

[0032] Figure 3 This indicates that the effect of the peptide of formula II on the body weight of HFD mice ( Figure 3 a) and food intake ( Figure 3 The impact of b).

[0033] Figure 4 This demonstrates the effects of the formula II peptide on anxiety and depression in mice, among which... Figure 4 a represents the dwell time in the open field test center area. Figure 4 b represents the number of times the test site remained in the central area of ​​the open field test. Figure 4 c represents the dwell time in the open-arm region of the high-cost cross maze experiment. Figure 4 d represents the stationary time during the tail suspension test.

[0034] Figure 5 This explains the GTT ( Figure 5 a) and ITT Figure 5 b) Effect of the formula II polypeptide on blood glucose in HFD mice during the experiment.

[0035] Figure 6 This study demonstrated the effect of peptide II on fatty liver in the Oil Red O staining assay. Figure 6 a represents Oil Red O staining of the liver. Figure 6 b represents the quantitative determination of liver Oil Red O staining.

[0036] Figure 7 This demonstrates the effect of formula II polypeptide on muscle in the HE staining experiment, in which... Figure 7 a is HE staining of muscle. Figure 7 b represents the quantification of muscle HE.

[0037] Figure 8 This diagram illustrates the grouping pattern of mice in Example 5. Detailed Implementation

[0038] The following description illustrates exemplary embodiments of the invention. It should be understood that such description is not intended to limit the scope of the invention, but is provided as a description of exemplary embodiments.

[0039] The term "PYY" used in this article 3-36 "Analog" refers to analogs or variants of the human polypeptide tyrosine (PYY), used to refer to polypeptides represented by Formula I and Formula II. 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] As used in this article, the term "amino acid" includes compounds represented by the following general formula:

[0041]

[0042] Wherein R and R' are discussed herein, unless otherwise stated, used alone or as part of another group, the term "amino acid" includes an amino group and a carboxyl group attached to the same carbon, referred to as the "α" carbon, wherein R and / or R' can be natural or non-natural side chains, including hydrogen. An absolute "S" configuration at the "α" carbon is generally referred to as "L" or "natural configuration." Where both "R" and the "R substituent" are hydrogen, the amino acid is glycine and is not chiral. Unless otherwise stated, the term "amino acid," used alone or as part of another group, includes, but is not limited to, natural or non-natural amino acids, where the carboxyl group is replaced (reduced) with methanol, such as valine, glycine, alanine, arylalanine, and heteroarylalanine.

[0043] The PYY of the present invention 3-36 Analogs can be prepared by chemical synthesis using various solid-state techniques. [14-16] .

[0044] The strategy employed in this invention is based on the Fmoc (9-fluorenylmethylmethoxycarbonyl) group for temporary protection of the α-amino group, combined with the tert-butyl group for temporary protection of the amino acid side chain. [14,15,17] .

[0045] The PYY of the present 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 using a THF solution of LiBH4.

[18] .

[0046] 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.

[0047] The PYY of the present invention 3-36The peptides in the analogue can be produced by a recombinant method, i.e., by culturing host cells containing the DNA sequence encoding the analogue and capable of expressing the peptide in a suitable nutrient medium under conditions that allow for peptide expression. Non-limiting examples of host cells suitable for expressing these peptides include *Escherichia coli*, *Saccharomycetes scerevisiae*, and mammalian BHK or CHO cell lines. In some embodiments, this fully recombinant fermentation step in the production process is desirable, for example, for economic reasons. [The text then abruptly shifts to a seemingly unrelated topic:] ...containing PYY... 3-36 The fusion protein inclusion bodies of the analog backbone were denatured and renatured to obtain a fusion protein with the correct conformation. After a series of treatments including enzyme digestion, sedimentation, and centrifugation, a high-content PYY protein was obtained. 3-36 The main chain of the analogue was purified by ion exchange chromatography to obtain PYY with high purity. 3-36 Analogous main chain.

[0048] The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. Excipients can be pharmaceutically acceptable inert substances, inactive substances, and / or substances that are not pharmaceutically active. The formulation of active pharmaceutical ingredients with different excipients is known in the art.

[19] Non-limiting examples of excipient functions include, but are not limited to, fillers, binders, lubricants, solvents, disintegrants, buffers, and preservatives. Those skilled in the art can easily select excipients with suitable functions based on the formulation; suitable carriers can be found in [reference needed].

[20] .

[0049] The terms "filler" include, but are not limited to, lactose, sucrose, etc.; "binder" includes, but is not limited to, starch, gelatin, etc.; "lubricant" includes, but is not limited to, magnesium stearate, etc.; "preservative" includes, but is not limited to, sodium benzoate, etc.; "disintegrant" includes, but is not limited to, methylcellulose, dry starch agar, etc.; "buffer" includes, but is not limited to, sodium chloride, etc.; and "solvent" includes, but is not limited to, ethanol, water, etc.

[0050] 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.

[0051] The term "NPYR-mediated disease" refers to the effects produced by binding to the NPYR receptor and activating downstream pathways. Because the NPYR receptor is widely expressed in the body, NPYR-mediated diseases include, but are 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, anxiety disorders, depression, and other mood disorders, as well as neurodegenerative diseases such as Alzheimer's disease.

[0052] The scope of this invention includes PYY 3-36 Similar substances and substances containing PYY 3-36 Compositions of analogues, wherein the composition comprises PYY as an active ingredient 3-36 Analogs and pharmaceutically acceptable analogs that can be used with PYY 3-36 Excipients used in combination. The PYY of this invention... 3-36 The analogue may be used alone or in combination with one or more other disease treatment agents for the treatment of related diseases; and may also be used in the preparation of medicaments for reducing food intake, reducing β-cell apoptosis, increasing pancreatic β-cell function, increasing β-cell clusters and / or restoring glucose sensitivity to β-cells.

[0053] The PYY of the present invention 3-36 Pharmaceutically, analogues can be administered in a variety of dosage forms (each including immediate-release, sustained-release, or controlled-release formulations), including but not limited to tablets, capsules, and injections. Those skilled in the art can select an appropriate dosage form based on the route of administration. These routes of administration include, but are not limited to, oral, transdermal, subcutaneous, intravenous, and intramuscular administration. Acceptable pharmaceutical procedures should be followed according to the prescribed routes of administration.

[21] The dosage forms were formulated based on the principles of pharmaceutical science. All dosage forms used were well-known to those skilled in the art and were administered with pharmaceutically acceptable excipients selected in accordance with the chosen route of administration and standard pharmaceutical practices.

[0054] The PYY of the present invention 3-36 Analogs or compositions can also be administered in combination with soluble polymers, such as those coupled to targeted drug carriers, biodegradable polymers for controlled drug release, and crosslinked or amphiphilic block copolymers of hydrogels.

[0055] 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.

[0056] Administration of the therapeutic agents of this 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 that can be used to treat or prevent a condition by administration of the composition of this invention. This dose is sufficient to exhibit a therapeutic, preventative, or ameliorative effect. Such effects include the treatment or prevention of the conditions listed herein. The precise effective amount for 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.

[0057] 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 as 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.

[0058] Abbreviations

[0059]

[0060]

[0061] Example 1. General method for chemical synthesis of polypeptide of formula I

[0062] IKP EAP GEX 11 ASP EEL X 18 X 19 Y YAX 23 LRH X 27 LN X 30 VT RQRY

[0063] Formula I

[0064] Without altering the secondary structure of Formula I, specific site substitutions of amino acids are achieved based on the principle of conserved amino acid substitution.

[0065] in,

[0066] X 11 Selected from aspartic acid, glycine, glutamic acid, and asparagine;

[0067] X 18 Selected from aspartic acid, glutamine, and histidine;

[0068] X 19 Selected from arginine and lysine;

[0069] X 23 Selected from serine, threonine, alanine, glycine, and methionine;

[0070] X 27 Selected from tyrosine, tryptophan, and phenylalanine;

[0071] X 30 It is selected from leucine, alanine, isoleucine, valine, and methionine.

[0072] Formula I of the present invention 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. Optionally, 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 (SASRINTM, Bachem Bioscience, Inc., King of Prussia, PA), and after the peptide sequence assembly is complete, the resulting peptide is released using a THF solution of LiBH4.

[18] .

[0073] Example 2. General method for chemical synthesis of formula II polypeptide (P1)

[0074] a. Resin swelling: Pour Fmoc-Ala-Wang resin into the reaction column, soak in DCM for 30 minutes, and then dry.

[0075] b. Deprotection: Add an appropriate amount of deprotection solution to the reaction column, purge with nitrogen and stir for 30 minutes, then dry under vacuum.

[0076] c. Weighing: Measure three molar amounts of the protected amino acids from the resin, and weigh three molar amounts of benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate (HBTU).

[0077] d. Deprotection washing: Add an appropriate amount of DMF to the reaction column, agitate with nitrogen for 2 minutes, dry the column, and repeat the operation 6 times.

[0078] e. Feeding: Add the prepared protected amino acids and HBTU to the reaction column, add 6 molar amounts of NMM resin, and stir with nitrogen for 30 minutes.

[0079] f. Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen for 2 minutes, drain, and repeat the operation 3 times.

[0080] g. Detection: Take an appropriate amount (10-20 beads) of resin in a small test tube, and add two drops each of solutions A, B, and C. Heat in a dry heater for 3 minutes (110℃). If the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete, and the next amino acid can be ligated. Repeat steps bf above until the last amino acid is ligated. (Solution A: 80% phenol + 20% anhydrous ethanol; Solution B: redistilled pyridine; Solution C: 5 g ninhydrin + 100 mL anhydrous ethanol)

[0081] h. Post-synthesis washing and drying: After the last amino acid is added and the protective layer has been removed, the mixture is dried under vacuum. An appropriate amount of methanol is added to the reaction column, and the column is agitated with nitrogen for 2 minutes. The mixture is then dried under vacuum again. An appropriate amount of DCM is added, and the column is agitated with nitrogen for 2 minutes. The mixture is then dried under vacuum again. This process is repeated 3 times. An appropriate amount of methanol is added to the reaction vessel, and the vessel is agitated with nitrogen for 2 minutes. The mixture is then dried under vacuum again. This process is repeated twice. The resin is then placed in a container and dried under vacuum in a desiccator for 12 hours.

[0082] i. Peptide digestion: Seal the centrifuge tubes and centrifuge at 4000 rpm for 3 minutes. Discard the supernatant, add ether, stir well, centrifuge again, and repeat the washing process 5 times. Place the peptides in a vacuum desiccator and dry for 24 hours to obtain a white powdery crude peptide product, which is then weighed.

[0083] Example 3. Cytotoxicity of Formula II polypeptide

[0084] HEK-293t cells were cultured in 96-well plates (37±0.5℃, 5±0.1% CO2). 0.16 mg of Formula II peptide was dissolved in 10 ml of PBS (4 μmol / mL), and then serially diluted (2 μmol / mL, 1 μmol / mL, 0.5 μmol / mL, 0.25 μmol / mL) before being added to high-glucose DMEM complete medium (Wuhan Saive Biotechnology Co., Ltd.). Toxicity was assessed using a CCK-8 assay kit (Wuhan Pronosei Life Sciences Co., Ltd.). Absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. IC50 curves were plotted using GraphPad Prism. Figure 1 ),

[0085] Based on the IC50 results of the peptide of formula II, the animal dosage was estimated according to the following formula:

[0086] Animal dosage (mg / kg) = Cellular dosage (μg / mL) × Fluid intake (mL / kg) ÷ Animal body weight (kg)

[0087] The converted animal dosage is 2356 μM / kg. In actual animal experiments, 80 μM / kg was used as the dosage (approximately 3% of 2356 μM / kg).

[0088] Example 4. Determination of Cyclic AMP

[0089] HEK-293t cells were cultured in 96-well plates and transfected with the recombinant expression plasmid pCAGIG containing the type II polypeptide using Lipo2000 transfection reagent (Sologopharm Technology, Beijing). After 24 hours of culture, the absorbance was measured at 450 nm using a cAMP kit (Sologopharm Technology, Beijing) and analyzed using GraphPad Prism.

[0090] The results show (e.g.) Figure 2 The cAMP levels in the formula II polypeptide group were reduced by approximately 5-40% compared to the model group, indicating that the formula II polypeptide can reduce intracellular cAMP levels by activating Gi / o protein and inhibiting adenylate cyclase. This is consistent with endogenous PYY. 3-36 The regulatory results are the same; therefore, the formula II polypeptide possesses endogenous PYY. 3-36 The activity.

[0091] Example 5. Effect of Formula II polypeptide on body weight

[0092] Thirty male C57BL / 6SPF grade 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%. The mice were divided into three groups (10 mice per group, e.g., ...). Figure 8 The study included a normal control group (normal feed and saline, NS), an HFD model group (high-fat diet and saline), and a formula II peptide administration group (high-fat diet and formula II peptide, P1). From days 1 to 7, an adaptive diet of 50% high-fat was administered for induction, and from days 8 to 50, a 100% high-fat diet was administered. From days 15 to 50, 0.9% saline or formula II peptide was administered subcutaneously daily, and daily body weight changes and total food intake 48 hours after each administration were measured.

[0093] Weight results as follows Figure 3 Starting from day 5, significant changes began to appear in the weight difference among the three groups of mice. The normal group, fed a standard diet, showed slow weight gain, with a weight difference of approximately 5-30% compared to the model group and approximately 5-15% compared to the Formula II peptide group. When fed a high-fat diet, the model group showed a weight difference of approximately 5-30% compared to the Formula II peptide group.

[0094] Food intake results as follows Figure 3 b. From day 6 to day 60, the food intake of the model group increased by about 5%-65% compared with the normal control group, while the food intake of the formula II polypeptide administration group decreased by 10%-60% compared with the model group.

[0095] Example 6. The anti-anxiety and antidepressant effects of Formula II peptides

[0096] Behavioral experiments were conducted on the three groups of mice in Example 5. The Open Field Test (OFT) (Pullman) involved placing mice in a test chamber and observing their activity during the test period. The Elevated Plus Maze Test (EPM) (Pullman) involved placing mice in the center of an elevated plus maze and observing their activity during the test period. The Tail Suspension Test (TST) involved suspending mice by their tails on a cage frame and observing their struggling, swinging, and stillness patterns while suspended.

[0097] In OFT and EPM experiments (e.g.) Figure 4 Compared with the control group, the model group mice showed significantly reduced movement time and frequency in the central region and reduced movement time in the open arm region; compared with the model group, the mice in the formula II polypeptide administration group showed significantly increased movement time and frequency in the central region and significantly increased movement time in the open arm region. In the TST experiment (e.g. Figure 4 d) The immobility time of mice in the model group was increased compared with that of mice in the control group, while the immobility time of mice in the formula II polypeptide administration group was significantly reduced compared with both the model group and the control group.

[0098] The results show that obesity exacerbates anxiety and depression in mice, and administration of the formula II peptide drug to mice with anxiety and depression significantly improves their anxiety and depression.

[0099] Example 7. Effect of Formula II polypeptide on blood glucose

[0100] Mice in Example 5 were fasted for one day, with normal water intake during this period. They were then administered 20% glucose by gavage, and blood glucose levels were measured using a glucometer and blood glucose test strips at 15 min, 30 min, 60 min, 90 min, and 120 min.

[0101] GTT results are as follows Figure 5 a. The blood glucose levels of mice in the model group increased significantly by 5-55% within 15-120 minutes compared to the control group; the blood glucose levels of the Formula II polypeptide administration group decreased by 5-55% within 15-120 minutes compared to the model group, showing that Formula II polypeptide has a significant effect on lowering blood glucose.

[0102] Example 8. Effect of Formula II polypeptide on insulin tolerance

[0103] Mice in Example 5 were fasted for one day, but had normal water intake during this period. Insulin solution of 0.75 U / kg was injected intraperitoneally, and blood glucose levels were measured using a glucometer and blood glucose test strips at 15 min, 30 min, 60 min, 90 min, and 120 min.

[0104] ITT results are as follows Figure 5 b. Within 120 minutes, blood glucose levels decreased by 5-90% in the control group, 5-50% in the model group, and 10-75% in the peptide II administration group. Compared with the model group, insulin tolerance was improved in the peptide II administration group.

[0105] Example 9. Effect of Formula II polypeptide on body fat accumulation

[0106] After anesthetizing the mice in Example 5, they were fixed supine on a mouse board, and the outer skin was dissected to expose subcutaneous fat. The accumulation of subcutaneous fat was recorded. Then, the organs were exposed, and the fat accumulation between organs was observed. Finally, the mouse muscle and liver tissues were collected and stored at -80°C.

[0107] The accumulation of subcutaneous and interorgan fat in the model group mice was 40-50% higher than that in the control group. The accumulation of Formula II peptide was 20-30% higher than that in the control group and 5-20% lower than that in the model group, indicating that the Formula II peptide group was significantly effective in reducing the accumulation of subcutaneous and interorgan fat.

[0108] Example 10. Effect of Formula II polypeptide on fatty liver

[0109] The mouse liver tissue collected in Example 9 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 Co., Ltd.) was diluted 3:2 (Oil Red O: distilled water) and stained. The sections were then mounted with glycerol gelatin and examined under a microscope.

[0110] The results are as follows Figure 6 In the model group, the number of fat droplets increased by 40%-60% compared to the control group, while the number of fat droplets in the formula II polypeptide administration group decreased by 20%-40% compared to the model group, indicating that the formula II polypeptide has a significant effect in reducing liver fat accumulation.

[0111] Example 11. Effects of Formula II polypeptide on muscle

[0112] The mouse muscle tissue collected in Example 9 was cryosectioned, stained with hematoxylin and eosin (Beijing Solarbio Science & Technology), mounted with glycerol gelatin, and examined under a microscope.

[0113] The results are as follows Figure 7The muscle area of ​​mice in the model group increased by 3-15% compared with that in the control group. The muscle area of ​​mice in the formula II polypeptide administration group was not significantly different from that in the model group, indicating that continuous administration of formula II polypeptide had no significant effect on muscle content in mice.

[0114] Example 12. Preparation of Formula II polypeptide drug formulation

[0115] A) Tablets

[0116] Tablets of the stated formulation specifications can be prepared according to standard tablet preparation procedures, for example, 100g of Formula II polypeptide, 0.2g of colloidal silica, 5g of magnesium stearate, 275g of microcrystalline cellulose, 11g of starch, and 98.8g of lactose. Appropriate coating can be used to improve palatability or delay absorption, and tablets can be prepared in 1000-tablet formulations.

[0117] B) Capsules

[0118] Capsules can be prepared according to conventional capsule preparation procedures to obtain the specified formulation specifications. For example, 100g of Formula II polypeptide, 150g of lactose, 50g of cellulose, 6g of magnesium stearate, and an appropriate amount of absorption enhancer can be filled into standard hard gelatin capsules to prepare 1000 capsules.

[0119] C) Injectable

[0120] Injectable preparations can be made using conventional procedures, such as preparing an injectable formulation with the following composition according to the methods described below.

[0121] Components quantity Formula II polypeptide 10g HCl or NaOH Adjust the pH value to 6-8. SBE-Cyclodextrin (Captisol) 55g Water for Injection Add to 5L Sodium chloride aqueous solution Appropriate amount

[0122] Dissolve the weighed Formula II polypeptide in a portion of water for injection at the optimal pH. Add SBE-cyclodextrin to the drug solution and stir for approximately 8 minutes. Adjust the pH to the desired value (between 6 and 8) by adding NaOH or HCl. Add water for injection and sodium chloride aqueous solution separately to make a final volume of 5 L and ensure isotonicity. Dispense into suitable ampoules to prepare 1000 injection vials. Before adjusting the pH, other inactive components such as solvents and cosolvents, solubilizers, antioxidants and reducing agents, preservatives, buffers, protectants, tension modifiers, and other special additives may be added as needed. The injection formulation must be sterile, pyrogen-free, and free of particulate matter in solution. It can be prepared by stirring in a pharmaceutically acceptable buffer, with or without cosolvents or other excipients. Before use, the solution should be isotonicized and sterilized with pharmaceutical-grade sodium chloride.

[0123] The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but this does not limit the scope of the invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the scope and essence of the invention should be within the scope of the invention.

[0124] References

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Claims

1. A type of PYY 3-36 Analogs, including the amino acid sequence shown in Formula I: IKP EAP GEX 11 ASP EEL X 18 X 19 Y YAX 23 LRH X 27 LN X 30 VT RQRY Formula I in, X 11 Selected from aspartic acid, glycine, glutamic acid, and asparagine; X 18 Selected from aspartic acid, glutamine, and histidine; X 19 Selected from arginine and lysine; X 23 Selected from serine, threonine, alanine, glycine, and methionine; X 27 Selected from tyrosine, tryptophan, and phenylalanine; X 30 It is selected from leucine, alanine, isoleucine, valine, and methionine.

2. A type of PYY 3-36 Analogs, including polypeptide sequences represented by Formula II: IKP EAP GED ASP EEL VRY YAG LRH WLN LVT RQRY(SEQ ID NO:1) Formula II.

3. A composition comprising the PYY as described in claim 1 or 2 3-36 Analogs and pharmaceutically acceptable excipients.

4. The PYY as described in claim 1 or 2 3-36 Use of analogues or the composition of claim 3 in the preparation of a medicament for treating diseases.

5. The use according to claim 4, characterized in that, The disease is an NPYR-mediated disease; preferably, the NPYR-mediated disease includes 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, kidney disease, asthma, chronic obstructive pulmonary disease, metabolic syndrome, anxiety disorders, depression and other mood disorders, and neurodegenerative diseases such as Alzheimer's disease.

6. A treatment method for an NPYR-mediated disease, comprising administering a therapeutically effective amount of the PYR as described in claim 1 or 2 to a subject in need. 3-36 The analogue or the composition of claim 3; preferably, the NPYR-mediated diseases include obesity and related complications, diabetes and related complications, glucose intolerance 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, kidney disease, asthma, chronic obstructive pulmonary disease, metabolic syndrome, anxiety disorders, depression and other mood disorders, and neurodegenerative diseases such as Alzheimer's disease.

7. The PYY as described in claim 1 or 2 3-36 The use of analogues or the composition of claim 3 in the preparation of medicaments for reducing food intake, increasing insulin sensitivity, and inhibiting fat accumulation.

8. The application according to claim 7, characterized in that, The fat accumulation includes subcutaneous fat accumulation, interorgan fat accumulation, and liver fat accumulation.

9. The PYY as described in claim 1 or 2 3-36 Use of analogues or the composition of claim 3 in the preparation of a medicament for treating anxiety and / or depression.

10. The PYY as described in claim 1 or 2 3-36 The use of analogues or the composition of claim 3 in the preparation of health products, nutritional supplements and as food additives.

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