Incretin analog and use thereof

Abstract

The present invention discloses an incretin analog and its use. The incretin analog can act on GLP-1 receptor, glucagon receptor and GIP receptor at the same time, and can exert the activities of GLP-1, glucagon and GIP at the same time. It not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effect of glucagon on body weight, energy metabolism and lipid metabolism, and also has the beneficial effect of GIP on sugar, lipid metabolism and appetite suppression, thereby producing a synergistic effect on sugar, lipid and energy metabolism. It has greater potential in being used in the preparation of drugs for treating metabolic syndrome, such as diabetes, dyslipidemia, fatty liver disease and obesity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an incretin analog and a use thereof. Background Art

[0002] Obesity is the result of long-term excess energy intake relative to consumption. It is a physiological dysfunction of the human body caused by environmental, genetic and endocrine reasons. In the past decade, obesity and its related diseases have become serious public health problems worldwide. It is worth noting that nearly 90% of patients with type 2 diabetes (T2DM) are overweight or obese. Studies have shown that the increase in the prevalence of obesity is positively correlated with the increase in the prevalence of T2DM. In addition, obesity can also lead to decreased glucose tolerance and aggravate T2DM-related metabolic abnormalities, including insulin resistance, dyslipidemia and hyperglycemia. Therefore, obesity makes the treatment of T2DM more difficult.

[0003] The efficacy of current drugs for treating obesity is relatively limited, and many drugs for treating obesity also have significant side effects, although some marketed glucagon-like peptide (GLP-1) receptor agonists have been used to treat obesity and T2DM, such as liraglutide and semaglutide. However, even semaglutide, which currently has the best weight loss effect, still cannot meet the high demand for weight loss in obese T2DM patients. Therefore, it is necessary to develop new and more effective anti-obesity and anti-diabetic drugs.

[0004] In recent years, an effective anti-obesity T2DM drug design strategy is to design single-molecule multi-target peptide drugs by rationally integrating the biological activities of endogenous gastrointestinal hormones in the human body to achieve efficient blood sugar reduction and weight loss. At present, this type of drug mainly focuses on the development of dual or triple agonists for the receptors corresponding to the three gastrointestinal hormones, GLP-1, glucagon, and glucose-dependent insulinotropic polypeptide (GIP), by rationally combining the physiological activities of these three gastrointestinal hormones. GLP-1 receptor, glucagon receptor and GIP receptor all belong to the GPCR receptor family, and from the perspective of sequence structure, GLP-1, glucagon and GIP have certain homology, making it possible to design triple receptor agonist peptide compounds for these three receptors. By acting on GLP-1 receptor, glucagon receptor and GIP receptor at the same time, the advantages of each receptor in blood sugar reduction or weight loss can be synergistically exerted, and the weight loss effect significantly improved compared with GLP-1 receptor agonists can be achieved. The difficulty in the development of such drugs is that since the compounds need to act on three receptors at the same time, the optimal ratio of the compounds' agonist activity for GLP-1 receptors, glucagon receptors and GIP receptors is still undetermined. It is generally believed that compounds need to have strong agonist activity for GLP-1 receptors, glucagon receptors and GIP receptors at the same time to achieve excellent hypoglycemic and weight loss effects (Cell Metabolism, 2022, 34, 1234–1247). Summary of the invention

[0005] The present invention provides an incretin analog and its use. The incretin analog can act on GLP-1 receptor, glucagon receptor and GIP receptor at the same time, and can exert the activities of GLP-1, glucagon and GIP at the same time. It not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effect of glucagon on body weight, energy metabolism and lipid metabolism, and also has the beneficial effect of GIP on sugar and lipid metabolism and appetite suppression, thereby producing a synergistic effect on sugar, lipid and energy metabolism. It has greater potential in being used in the preparation of drugs for treating metabolic syndrome, such as diabetes, dyslipidemia, fatty liver disease and obesity.

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

[0007] An incretin analog, wherein the amino acid sequence structure of the incretin analog is:

[0008]

[0009] (SEQ ID NO:1)

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

[0011] The present invention also provides a use of an incretin analog or a pharmaceutical composition of an incretin analog in preparing a medicament for treating metabolic diseases or conditions, including diabetes, dyslipidemia, fatty liver disease and obesity.

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

[0013] (1) The incretin analog of the present invention can act on the GLP-1 receptor, glucagon receptor and GIP receptor at the same time, and can exert the activities of GLP-1, glucagon and GIP at the same time; it not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effects of glucagon on body weight, energy metabolism and lipid metabolism, and also has the beneficial effects of GIP on sugar and lipid metabolism and appetite suppression, thereby producing a synergistic effect on sugar, lipid and energy metabolism; it has greater potential in the preparation of drugs for treating metabolic syndrome, such as diabetes, dyslipidemia, fatty liver disease and obesity;

[0014] (2) An incretin analog of the present invention has significantly better glucose-lowering and weight-reducing activities than tirzepatide and SAR441255;

[0015] (3) The incretin analog of the present invention has a different ratio of agonist activity for GLP-1 receptor, glucagon receptor and GIP receptor than similar drugs reported previously, but achieves better hypoglycemic and weight loss effects, achieving unexpected beneficial effects, and providing new ideas for the design of such drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The percentage of body weight change after long-term administration of the incretin analogs of the present invention and tirzepatide to DIO mice for 21 days.

[0017] Figure 2 The blood glucose curves of the glucose tolerance test conducted on DIO mice after long-term administration of the incretin analogs of the present invention and tirzepatide for 21 days.

[0018] Figure 3 It is the percentage change in body weight of the incretin analogs of the present invention and SAR441255 in DIO mice after long-term administration for 21 days.

[0019] Figure 4This is the blood glucose curve of the glucose tolerance test conducted on DIO mice after long-term administration of the incretin analog of the present invention and SAR441255 for 21 days. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0021] 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.

[0022] Furthermore, the amino acids mentioned in the present invention can be abbreviated as follows according to the nomenclature of IUPAC-IUB:

[0023] Alanine (Ala, A); valine (Val, V); phenylalanine (Phe, F); asparagine (Asn, N); threonine (Thr, T); cysteine ​​(Cys, C); arginine (Arg, R); glutamine (Gln, Q); glycine (Gly, G); histidine (His, H); aspartic acid (Asp, D); isoleucine (Ile, I); glutamic acid (Glu, E); leucine (Leu, L); tyrosine (Tyr, Y); proline (Pro, P); methionine (Met, M); serine (Ser, S); tryptophan (Trp, W); lysine (Lys, K).

[0024] Furthermore, unless explicitly stated otherwise, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0025] Further, the "-NH2" moiety at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0026] Furthermore, in addition to natural amino acids, two unnatural amino acids, dextrorotatory alanine (dAla) and α-aminoisobutyric acid (Aib), are used in the sequences of the present invention.

[0027] 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.

[0028] Example 1

[0029] Synthesis of SEQ ID NO:1

[0030]

[0031] In a manual polypeptide synthesis tube, Fmoc-RinkAmide-MBHA resin was used for polypeptide synthesis, the resin loading was 0.312mmol / g, and the synthesis scale was 0.1mmol. First, 0.32g of resin was weighed and placed in a polypeptide synthesis tube, and the resin was washed 3 times with 7mL of dichloromethane for 5 minutes each time, and then the resin was swollen with 7mL of dichloromethane for 30 minutes. After filtering out the dichloromethane, the resin was washed 3 times with 7mL of DMF to complete the resin swelling step. Then the Fmoc protecting group on the resin was removed, 7mL of 20% piperidine / DMF solution was added, nitrogen was bubbled for half an hour, the piperidine solution was filtered out, and the resin was washed 4 times with 7mL of DMF to complete the Fmoc protecting group removal step. Next, add the first amino acid Fmoc-Lys(Boc)-OH, and use HBTU / HOBT / DIPEA condensing agent (0.4eq, 0.4eq, 0.8eq) for amino acid coupling. Nitrogen bubbling is used for 1.5 hours in the coupling step. After the coupling is completed, the resin is washed 3 times with 7mL DMF to complete the amino acid coupling step. Next, according to the peptide sequence, the corresponding amino acids are added in turn for Fmoc deprotection and amino acid coupling steps to complete the synthesis of the main peptide chain. At the fatty acid modification site of the polypeptide, Fmoc-Lys(Dde)-OH is used for Lys at position 14. After the polypeptide peptide chain synthesis is completed, 7mL 2% hydrazine hydrate / DMF (v / v) is added and bubbled for half an hour to remove the Dde protecting group. Then, according to the structure of the side chain, Fmoc-Glu-OtBu was added respectively, and HBTU / HOBT / DIPEA condensation agent was used for coupling for 1.5 hours. After the reaction, 7 mL of 20% piperidine / DMF solution was used to remove the Fmoc protecting group, and then Fmoc-Glu-OtBu was added again, and HBTU / HOBT / DIPEA condensation agent was used for coupling for 1.5 hours. After the reaction, 7 mL of 20% piperidine / DMF solution was used to remove the Fmoc protecting group. Finally, palmitic acid was added, and HBTU / HOBT / DIPEA condensation agent was used for coupling for 1.5 hours to complete the synthesis of the compound.

[0032] Finally, the obtained resin with the polypeptide was transferred to a round-bottom bottle, and the resin was cut with 5 mL of cutting agent Reagent R (TFA / thioanisole / phenol / EDT, 90:5:3:2, V / V), and the reaction was kept at a constant temperature of 30°C in an oil bath for 2 h. The cutting solution was poured into 40 mL of ice ether, and the crude product was washed 3 times with 15 mL of ice ether after refrigerated centrifugation, and finally dried with nitrogen to obtain a crude peptide. The crude peptide was purified using a preparative reverse phase HPLC system (C18 reverse phase preparative column, 250 mm × 20 mm, 12 μm), mobile phase A: 0.1% TFA / water (V / V), mobile phase B: methanol (V / V); flow rate was 8 mL / min; detection wavelength was 214 nm. A linear gradient (20% B ~ 70% B / 30 min) was used for elution, the target peak was collected, and the pure product was obtained by freeze drying after removing methanol. The purity was greater than 98%, and the molecular weight of the target polypeptide was confirmed by MS. Theoretical relative molecular mass is 4895.6. ESI-MS m / z: calculated value [M+3H] 3+ 1632.9,[M+4H] 4+ 1224.9; observed value [M+3H] 3+ 1632.5,[M+4H] 4+ 1224.7.

[0033] Example 2

[0034] Determination of the agonist activity of incretin analogs on GLP-1 receptor, glucagon receptor and GIP receptor

[0035] HEK293 or CHO cell lines stably expressing GLP-1 receptor, glucagon receptor or GIP receptor were cultured in DMEM + 100μg / mL Hygromycin B medium containing 10% fetal bovine serum at 37℃ and 5% carbon dioxide concentration. To maintain the physiological activity of the cells, the experimental cell confluence was controlled at about 80%. The compound was diluted with DMSO in a gradient, and then diluted with 1×Stimulation Buffer in a 10-fold gradient. The stable transfected cells were cultured to 80% confluence, digested with Cell Dissociation Buffer solution, and collected. After counting, the cells were inoculated at 9μL / well in a 384-well plate. 1μL of the diluted compound was added to the corresponding experimental well, centrifuged and incubated at 37℃ for 30min. Eu-cAMP was diluted to a working concentration with Detection buffer, and 5μL / well was added to the corresponding experimental well. Dilute ULight-anti-cAMP to the working concentration with Detection buffer, then take 5 μL / well and add it to the corresponding experimental wells; incubate at room temperature for 1 hour after centrifugation. After incubation, use Perkin Elmer multifunctional microplate reader to detect the readings at 665nm and 620nm. Plot the ratio (665 / 620) against the compound concentration, and use GraphPad Prism software nonlinear regression method to perform curve fitting and EC 50 calculate.

[0036] Table 1: EC values ​​of incretin analogs for human GLP-1 receptor, glucagon receptor and GIP receptor 50 Value (expressed in pM)

[0037]

[0038] As shown in Table 1, the incretin analogs of the present invention have a stimulatory activity on the human GLP-1 receptor that is about 3.5 times stronger than that of natural GLP-1; the stimulatory activity on the human GIP receptor is about 1.9 times weaker than that of natural GIP; and the stimulatory activity on the human glucagon receptor is about 20.5 times weaker than that of natural glucagon. Compared with similar drugs reported so far (Cell Metabolism, 2022, 34, 1–16; Cell Metabolism, 2022, 34, 1234–1247), the incretin analogs of the present invention have strong GLP-1 receptor stimulatory activity, strong GIP receptor stimulatory activity and weaker glucagon receptor stimulatory activity. The ratio of their stimulatory activity on the GLP-1 receptor, glucagon receptor and GIP receptor is significantly different from that of similar drugs reported so far, and they have a special ratio of stimulatory activity on the GLP-1 receptor, glucagon receptor and GIP receptor.

[0039] Example 3

[0040] Effects of incretin analogs on body weight, blood glucose and blood lipids in diet-induced obese (DIO) mice

[0041] Male C57BL / 6J mice, weighing about 22 g, were fed a high-fat diet D12492 from Research Diets for 18 weeks to establish the DIO mouse model. DIO mice weighing 46–54 g were selected for the experiment, and animals were assigned to different treatment groups based on body weight to ensure uniform initial body weight in each group. To determine food intake in each group of mice, DIO mice were housed in single cages, with 8 mice in each group. There were 6 groups in the experiment, including blank control (saline), tirzepatide (30 nmol / kg), and SEQ ID NO: 1 (30 nmol / kg); and blank control (saline), SAR441255 (30 nmol / kg), and SEQ ID NO: 1 (30 nmol / kg). The injection frequency was once a day (saline, SAR441255, and SEQ ID NO: 1) or once every two days (tirzepatide), and the administration lasted for 21 days, with each injection volume of 5 mL / kg. The weight of mice was recorded every day, and an oral glucose tolerance test (OGTT) was performed on the 22nd day after the 21-day treatment experiment. After an 8-hour overnight fast, DIO mice were orally given 1.5 g / kg of glucose. Blood glucose levels were then measured at different time points (0, 15, 30, 60, and 120 minutes) to assess 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 triglyceride (TG) and total cholesterol (TC) levels were measured.

[0042] Table 2: Changes in body weight and glucose tolerance of DIO mice during the 3-week dosing period

[0043]

[0044] *** : Compared with the blank control group, P<0.001; # : P<0.05 compared with tirzepatide group; ### : P<0.001 compared with the tirzepatide group. The results are expressed as the mean±SD of 8 mice in each group.

[0045] like Figure 1 , Figure 2 The results in Table 2 show that the incretin 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 that of the GLP-1 / GIP receptor dual agonist tirzepatide that has been marketed. In addition, the OGTT blood sugar value and blood sugar AUC value after the treatment show that the effect of the incretin analogs of the present invention on improving the glucose tolerance of DIO mice is also significantly better than that of tirzepatide, indicating that the incretin analogs of the present invention have excellent hypoglycemic and weight loss effects.

[0046] Table 3: Changes in body weight and glucose tolerance of DIO mice during the 3-week dosing period

[0047]

[0048] *** : Compared with the blank control group, P<0.001; ### : P<0.001 compared with SAR441255 group. The results are expressed as the mean±SD of 8 mice in each group.

[0049] like Figure 3 , Figure 4The results in Table 3 show that the incretin analogs of the present invention can significantly reduce the body weight of DIO mice after continuous administration for 21 days in DIO mice, and their weight loss effect is significantly better than that of the same type of clinical drug SAR441255. In addition, the OGTT blood glucose values ​​and blood glucose AUC values ​​after the end of treatment show that the incretin analogs of the present invention are also significantly better than SAR441255 in improving the glucose tolerance of DIO mice, indicating that the incretin analogs of the present invention have excellent hypoglycemic and weight loss effects. It is worth noting that compared with the reported in vitro GLP-1 receptor, glucagon receptor and GIP receptor agonist activity of SAR441255 (Cell Metabolism, 2022, 34, 1–16), the incretin analogs of the present invention have stronger GLP-1 receptor agonist activity, similar GIP receptor agonist activity and significantly weaker glucagon receptor agonist activity, but the incretin analogs of the present invention show significantly better hypoglycemic and weight loss effects.

[0050] Table 4: Serum triglyceride (TG) and total cholesterol (TC) levels in DIO mice after 3 weeks of treatment

[0051] Sample (dose) Total cholesterol (mmol / L) Triglyceride (mmol / L) Blank control (normal saline group) 8.26±0.62 1.22±0.11 Tirzepatide (30nmol / kg) <![CDATA[6.35±0.54 *** ]]> <![CDATA[0.71±0.08 *** ]]> SEQ ID NO: 1 (30 nmol / kg) <![CDATA[4.32±0.29 ***,### ]]> <![CDATA[0.38±0.05 ***,### ]]>

[0052] *** : 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 8 mice in each group.

[0053] Table 5: Serum triglyceride (TG) and cholesterol (TC) levels in DIO mice after 3 weeks of treatment

[0054] Sample (dose) Cholesterol (mmol / L) Triglyceride (mmol / L) Blank control (normal saline group) 7.93±0.48 1.14±0.09 SAR441255 (30nmol / kg) <![CDATA[5.86±0.51 *** ]]> <![CDATA[0.54±0.07 *** ]]> SEQ ID NO: 1 (30 nmol / kg) <![CDATA[4.55±0.32 ***,### ]]> <![CDATA[0.41±0.06 ***,# ]]>

[0055] *** : Compared with the blank control group, P<0.001; # : P<0.05 compared with SAR441255 group; ### : P<0.001 compared with SAR441255 group. The results are expressed as the mean±SD of 8 mice in each group.

[0056] As shown in the results of Tables 4 and 5, continuous administration of the incretin analogs of the present invention to DIO mice for 21 days can significantly reduce the serum triglyceride and cholesterol levels of the mice, and the effect of the incretin analogs of the present invention on reducing serum lipid levels is significantly stronger than that of tirzepatide and SAR441255.

Claims

1. An incretin analog, characterized in that: The amino acid sequence structure of the incretin analog is: 。 2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the incretin analogue according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

3. Use of the incretin analogue according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating a metabolic disease or condition; the metabolic disease or condition is diabetes, dyslipidemia, fatty liver disease or obesity.

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