Long-acting polypeptide analogue and application thereof

Abstract

The invention discloses a long-acting polypeptide analogue and application thereof, the polypeptide analogue can act on a GLP-1 receptor, a GIP receptor and a Y2 receptor at the same time, and can play the biological activities of GLP-1, GIP and PYY3-36 at the same time; the PYY3-36 GLP-1 has the treatment effect of GLP-1 on diabetes mellitus, also has the beneficial effect of GIP on glucose and lipid metabolism and appetite inhibition, and also has the beneficial effect of PYY3-36 on appetite inhibition, so that the PYY3-36 GLP-1 has a synergistic effect on glucose, lipid and energy metabolism; the application of the compound in preparation of drugs for treating metabolic syndromes, such as diabetes, obesity and dyslipidemia, has greater potential in preparation of drugs for treating diseases such as diabetes, obesity and dyslipidemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a long-acting polypeptide analog and use thereof. Background Art

[0002] The number of obese patients has increased dramatically in recent years and has become a major public health crisis. Obesity is a major risk factor for multiple diseases, including cardiovascular disease and type 2 diabetes mellitus (T2DM). Many international professional organizations have classified obesity as a chronic disease due to the widespread and severe morbidity caused by this disease. Currently, the most effective therapeutic intervention for obesity is gastrointestinal diversion surgery, specifically Roux-en-Y gastric bypass. It is widely accepted that the secretion of many gut hormones, including glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), is altered after such surgery. These changes in gut hormones are thought to play an important role in the observed enhancement of glycemic control and weight loss. Despite its curative potential, gastrointestinal diversion surgery remains an invasive procedure with surgical risks.

[0003] GLP-1 receptor agonists have been shown to reduce weight, improve multiple cardiovascular risk parameters and glycemic control. Therefore, GLP-1 receptor agonists have become a successful treatment option for T2DM, and several long-acting GLP-1 receptor agonists (liraglutide, semaglutide, dulaglutide, etc.) have been developed, which have better efficacy than natural GLP-1, which has a short half-life (several minutes) due to renal clearance and rapid dipeptidyl peptidase-IV cleavage. PYY 1-36 Secreted from intestinal L cells simultaneously with GLP-1 and rapidly cleaved into PYY by DPP-IV after eating 3-36 , which has better selectivity for the Y2 receptor than for other NPY receptors (Y1 receptor, Y4 receptor, and Y5 receptor). 3-36 Long-acting PYY has been reported to reduce food intake via the Y2 receptor 3-36 Analogues compared to PYY 3-36 It has a better weight loss effect. In addition, PYY has been reported in animal models 3-36 The improved efficacy was shown when co-administered with exendin-4, oxyntomodulin, or GLP-1, indicating the great potential of designing single-molecule peptides with dual activation potency on GLP-1 receptor and Y2 receptor.

[0004] Glucose-dependent insulinotropic polypeptide (GIP) is an incretin that is secreted by K cells in the upper small intestine in response to food stimulation. Under normal physiological conditions, the level of GIP after a meal is about 4 times that of GLP-1. GIP accounts for most of the incretin effect of insulin secretion in the human body, and has important additional functions that are different from GLP-1. Unlike GLP-1, GIP can promote both glucagon and insulin secretion in a blood sugar-dependent manner, and it stimulates glucagon secretion in a dose-dependent manner under hypoglycemic conditions and stimulates insulin secretion under hyperglycemic conditions. By integrating the biological activities of GLP-1 and GIP, the GLP-1 / GIP receptor dual agonist designed achieves better blood sugar control and weight loss effects than the GLP-1 receptor agonist. If PYY can be further integrated 3-36 The biological activity of the GLP-1 / GIP receptor dual agonist is integrated to design a GLP-1, GIP and PYY 3-36 The triple biologically active GLP-1 / GIP / Y2 receptor triple agonist peptide analogue can not only use the activity of GLP-1 and GIP to achieve efficient glucose reduction and effective weight loss, but also use PYY 3-36 The appetite suppressant effect achieves a better weight loss effect. Summary of the invention

[0005] The present invention provides a long-acting polypeptide analog and its use, wherein the polypeptide analog can act on GLP-1 receptor, GIP receptor and Y2 receptor at the same time, and can exert the effects of GLP-1, GIP and PYY at the same time. 3-36 It has biological activity; it not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effect of GIP on sugar and lipid metabolism and appetite suppression, and also has PYY 3-36 It has a beneficial effect on appetite suppression, thereby producing a synergistic effect on sugar, fat and energy metabolism; it has greater potential for use in the preparation of drugs for the treatment of metabolic syndrome, diseases such as diabetes, obesity and dyslipidemia.

[0006] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] A long-acting polypeptide analog, the amino acid sequence structure of the polypeptide analog is:

[0008]

[0009] (SEQ ID NO:1)

[0010] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned long-acting polypeptide analog and a pharmaceutically acceptable carrier, diluent or excipient.

[0011] The present invention also provides a use of a long-acting polypeptide analog or a pharmaceutical composition of a long-acting polypeptide analog in preparing a drug for treating metabolic diseases or conditions, including diabetes, obesity and dyslipidemia.

[0012] Compared with the prior art, the beneficial technical effects of the present invention are:

[0013] (1) The long-acting polypeptide analog of the present invention can act on the GLP-1 receptor, GIP receptor and Y2 receptor at the same time, and can exert the effects of GLP-1, GIP and PYY at the same time. 3-36 It has biological activity; it not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effect of GIP on sugar and lipid metabolism and appetite suppression, and also has PYY 3-36 The beneficial effect of suppressing appetite, thus producing a synergistic effect on sugar, fat and energy metabolism; it has greater potential for application in the preparation of drugs for the treatment of metabolic syndrome, such as diabetes, obesity and dyslipidemia;

[0014] (2) The sequence fragment (RHYLN WLTRQ RY) at positions 31-42 in the sequence structure of the long-acting polypeptide analog of the present invention is a Y2 receptor agonist fragment; short-chain Y2 receptor agonist fragments are usually difficult to achieve strong Y2 receptor agonist activity, but the short-chain Y2 receptor agonist fragment in the long-acting polypeptide analog of the present invention not only achieves the strong agonist activity of the polypeptide analog on the Y2 receptor, but also achieves the selective agonist activity of the polypeptide analog on the Y2 receptor, achieving an unexpected effect;

[0015] (3) The long-acting polypeptide analogs of the present invention have significantly better hypoglycemic, weight-reducing and lipid-regulating activities than tirzepatide;

[0016] (4) The long-acting peptide analogs of the present invention successfully integrate GLP-1, GIP and PYY through simple linear peptide design methods 3-36 The biological activities of these three peptides with huge structural differences achieve better blood sugar lowering and weight loss effects, and provide new ideas for the design of such drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the percentage change in body weight of the polypeptide analog of the present invention in DIO mice after long-term administration for 21 days.

[0018] Figure 2 This is the blood glucose curve of a glucose tolerance test conducted on DIO mice after long-term administration of the polypeptide analog of the present invention for 21 days. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0020] Unless otherwise defined herein, the scientific and technical terms used in the specification shall have the meanings commonly understood by those of ordinary skill in the art.

[0021] Furthermore, the amino acids mentioned in the present invention can be abbreviated as follows according to the nomenclature of IUPAC-IUB:

[0022] Cysteine ​​(Cys, C); Phenylalanine (Phe, F); Alanine (Ala, A); Glycine (Gly, G); Glutamic acid (Glu, E); Glutamine (Gln, Q); Methionine (Met, M); Arginine (Arg, R); Lysine (Lys, K); Tyrosine (Tyr, Y); Leucine (Leu, L); Proline (Pro, P); Tryptophan (Trp, W); Serine (Ser, S); Threonine (Thr, T); Aspartic acid (Asp, D); Asparagine (Asn, N); Valine (Val, V); Isoleucine (Ile, I); Histidine (His, H).

[0023] Furthermore, unless explicitly stated otherwise, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0024] Further, the "-NH2" moiety at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0025] Furthermore, in addition to natural amino acids, the sequences of the present invention also use a non-natural amino acid, α-aminoisobutyric acid (Aib).

[0026] In order to illustrate the present invention in more detail, this specification provides the following specific implementation schemes, but the solutions of the present invention are not limited thereto.

[0027] Example 1

[0028] Synthesis of SEQ ID NO:1

[0029]

[0030] The synthesis method of peptide analogs adopts the resin Fmoc / tBu orthogonal protection solid phase synthesis strategy, and uses Fmoc-RinkAmide-MBHA resin with a loading amount of 0.356mmol / g as the solid phase carrier of the peptide. 0.281g of resin was added to the synthesis device, and 7mL of DMF was added to wash the resin once, and the DMF was filtered out using a vacuum pump. 7mL of dichloromethane was added to the reaction device to swell the resin for 5 minutes. After completion, the reaction solution was filtered out, and 7mL of dichloromethane was added again. Swell for 30 minutes, and after filtration, 7mL of DMF was added to wash the resin 3 times, each for 1 minute. After the resin swells, 7mL of 20% piperidine / DMF solution needs to be added to remove the Fmoc protecting group on the amino group of the resin. Nitrogen is passed into the device to fully swell the resin in the solution and react for 30 minutes. After the deprotection reaction is completed, the reaction solution is filtered out, and 7mL of DMF is added to wash the resin for 1 minute each time, and the operation is repeated 4 times. Weigh the first amino acid Fmoc-Tyr(tBu)-OH, add condensing agent (HBTU / HOBT / DIPEA) to dissolve and pre-activate, then add to the reaction device, introduce nitrogen to react for 1.5 hours, filter out the reaction solution after the reaction, add 7mL DMF to wash the resin 3 times, 1 minute each time. Next, remove the Fmoc protecting group of the α-amino group of the amino acid, and then add amino acids one by one from the C-terminus to the N-terminus according to the sequence, repeat the condensation and Fmoc deprotection reaction until the N-terminal Boc-Tyr(tBu)-OH condensation is completed, wherein Fmoc-Lys(Dde)-OH is used for Lys at position 20. For fatty acid side chain modification, the ε-amino protecting group of Lys at position 20 is Dde, and the deprotection reaction system used is 2% hydrazine hydrate / DMF solution. After the Dde protecting group was removed, the side chain modifiers Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu and octadecane dioic acid mono-tert-butyl ester were added in sequence. The steps of condensation reaction and Fmoc deprotection reaction were the same as those of the main chain. The final resin was washed 3 times with 7 mL of dichloromethane and dried. The cutting solution used for separation of resin and peptide was TFA / thioanisole / phenol / EDT (90:5:3:2, V / V). The dried resin filter cake was transferred to a 10 mL reaction vial, 5 mL of cutting solution was added, and the reaction was shaken at room temperature for 3 hours. The reaction solution was filtered into ten times the volume of ice ether. After centrifugation, the precipitate was washed 3 times with ice ether and dried at room temperature to obtain the crude peptide. The crude peptide was purified using preparative HPLC: pump A: 0.1% TFA / water (V / V), pump B: methanol (V / V); flow rate was 6 mL / min; detection wavelength was 214 nm, gradient elution (20% B-70% B / 60 min), target peaks were collected, methanol was removed by rotary evaporation, and pure peptide was collected by freeze drying. Analytical HPLC verified that the purity was greater than 98%, and MS mass spectrometry confirmed that the molecular weight was the target polypeptide.Theoretical relative molecular mass is 5663.5. ESI-MS m / z: calculated value [M+3H]. 3+ 1888.8,[M+4H] 4+ 1416.9; observed value [M+3H] 3+ 1888.2,[M+4H] 4+ 1416.2.

[0031] Example 2

[0032] Determination of the agonist activity of peptide analogs on GLP-1 receptor, GIP receptor and four NPY receptors

[0033] Functional assays of peptide analogs for agonism of GLP-1 and GIP receptors were performed by measuring the cAMP response of HEK293 cells stably expressing human GLP-1 and GIP receptors. Compounds were diluted 4-fold in DMSO at 10 points, and the cell suspension was transferred to a 15 mL conical tube containing 10 mL of HBSS and centrifuged at 1000 rpm for 5 minutes at room temperature to sediment the cells. The cell pellet was then resuspended in 10 mL of HBSS, and the cell concentration and cell viability were counted on a Vi-cell. HEK293 cells expressing GLP-1 and GIP receptors were resuspended in assay buffer at a concentration of 200,000 cells / mL, and 10 μL of cells were transferred to an OptiPlate-384 plate. 100 nL of compound was transferred to the OptiPlate-384 plate using an Echo, and the plate was centrifuged at 1000 rpm for 5 seconds. Then, the plates were incubated at room temperature for 30 minutes, the detection reagent was added, and the 384-well plates were covered with TopSeal-A film and incubated at room temperature for 60 minutes. Finally, the TopSeal-A film was removed, the data were read using EnVision, and the EC was determined using GraphPad Prism 7.0. 50 Numeric value.

[0034] HEK-293 cells stably expressing human NPY receptors (Y1 receptor, Y2 receptor, Y4 receptor or Y5 receptor) and Gα15 protein were used to determine the agonistic effect of peptide compounds on NPY receptors. Cells were grown in culture medium, and 20 μL of cell suspension was added to each well of a 384-well plate (20,000 cells per well) and incubated overnight at 37°C and 5% CO2. Then, the cell plate was removed from the incubator, the culture medium was discarded, and 20 μL of FLIPR assay buffer and 20 μL of 2x concentration of Fluo-4 Direct loading buffer were added. The cell plate was incubated at 37°C and 5% CO2 for 50 minutes and then incubated at room temperature for 10 minutes. The peptide compound to be tested was diluted 4-fold at 10 points, and 750 nL of the peptide compound was transferred to a 384-well plate using Echo. Then, 30 μL of FLIPR assay buffer was added to the 384-well plate. Finally, the cell plate and compound plate were placed in the FLIPR Tetra system (Molecular Devices), 10 μL of the tested peptide was transferred to the cell plate, and the fluorescence signal (Ex 494 nm / Em 516 nm) was measured. 50 Data were analyzed using GraphPad Prism 7.0.

[0035] Table 1: EC values ​​of peptide analogs for GLP-1 receptor, GIP receptor and four NPY receptors 50 Value (in nM)

[0036]

[0037] As shown in Table 1, the polypeptide analogs of the present invention have strong agonist activity on GLP-1 receptor, GIP receptor and Y2 receptor. In addition, compared with other NPY receptors (Y1 receptor, Y4 receptor and Y5 receptor), the polypeptide analogs of the present invention also have excellent selective agonist activity on Y2 receptor, and their agonist selectivity for Y2 receptor is significantly better than that for PYY. 3-36 The selective activation of the Y2 receptor by the polypeptide analogs of the present invention can minimize side effects, such as increased appetite caused by activation of the Y1 receptor and the Y5 receptor, and additional inhibition of gallbladder emptying and pancreatic enzyme secretion caused by activation of the Y4 receptor (J. Med. Chem. 2018, 61, 23, 10519–10530).

[0038] Example 3

[0039] Effects of peptide analogs on body weight, blood glucose and blood lipids in diet-induced obese (DIO) mice

[0040] 20g male C57BL / 6J mice were selected and fed with high-fat diet (D12492, Research Diets) for more than 16 weeks to create a DIO mouse model. The experiment selected DIO mice weighing more than 45 grams, and the initial weight of each group of DIO mice was kept similar. There were 6 DIO mice in each group. The blank group was given normal saline (10mL / kg), and the drug-treated groups were given tirzepatide (30nmol / kg) and SEQ ID NO: 1 (30nmol / kg), respectively. The drug-treated period was subcutaneous injection once every 2 days. The weight of the mice was recorded every two days, and an oral glucose tolerance test (OGTT) was performed on the 22nd day of the experiment. The DIO mice fasted for 8 hours at night and then orally administered 1.5g / kg of glucose. Blood glucose levels were then measured at different time points (0, 15, 30, 60 and 120 minutes) to evaluate changes in glucose tolerance. After completing the OGTT, each group of DIO mice rested for one day. On the 23rd day, blood samples were collected from each group of DIO mice in a non-fasting state to prepare serum, and the serum triglyceride and cholesterol levels were measured.

[0041] Table 2: Changes in body weight and glucose tolerance of DIO mice during the 3-week dosing period

[0042]

[0043] *** : Compared with the blank control group, P<0.001; ### : P<0.001 compared with the tirzepatide group. The results are expressed as the mean±SD of 6 mice in each group.

[0044] like Figure 1 , Figure 2 The results in Table 2 show that the polypeptide analogs of the present invention can significantly reduce the body weight of DIO mice after continuous administration for 21 days, and their weight loss effect is significantly better than tirzepatide, indicating that the additional Y2 receptor agonist activity significantly improves the weight loss effect of the polypeptide analogs of the present invention. In addition, the OGTT blood sugar values ​​and blood sugar AUC values ​​after the treatment show that the effect of the polypeptide analogs of the present invention on improving the glucose tolerance of DIO mice is also significantly better than tirzepatide, indicating that the polypeptide analogs of the present invention have excellent hypoglycemic and weight loss effects.

[0045] Table 3: Serum triglyceride and cholesterol levels in DIO mice after 3 weeks of treatment

[0046] Sample (dose) Cholesterol (mmol / L) Triglyceride (mmol / L) Blank control (normal saline group) 11.12±0.85 1.51±0.13 Tirzepatide (30 nmol / kg) <![CDATA[7.23±0.61 *** ]]> <![CDATA[0.76±0.05 *** ]]> SEQ ID NO: 1 (30 nmol / kg) <![CDATA[4.68±0.33 ***,### ]]> <![CDATA[0.41±0.02 ***,### ]]>

[0047] *** : Compared with the blank control group, P<0.001; ###: P<0.001 compared with the tirzepatide group. The results are expressed as the mean±SD of 6 mice in each group.

[0048] As shown in the results of Table 3, the polypeptide analogs of the present invention can significantly reduce the serum triglyceride and cholesterol levels of DIO mice after continuous administration for 21 days, and the effect of the polypeptide analogs of the present invention on reducing serum lipid levels is significantly stronger than that of tirzepatide.

Claims

1. A long-acting polypeptide analog, characterized in that: The amino acid sequence structure of the polypeptide analog is:

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide analog according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

3. Use of a polypeptide analogue according to claim 1 or a pharmaceutical composition according to claim 2 in the preparation of a medicament for treating a metabolic disease or condition.

4. The use according to claim 3, characterized in that The metabolic diseases or conditions are diabetes, obesity and dyslipidemia.

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