Intestinal insulinotropic analogue with multiple biological activities, synthetic method, pharmaceutical composition and application

Abstract

The invention relates to the technical field of biological medicine, and discloses an intestinal insulinotropic analogue with multiple biological activities, a synthesis method, a pharmaceutical composition and application, the synthesized intestinal insulinotropic analogue has triple agonist activity of a GLP-1 receptor, a glucagon receptor and a CCK-1 receptor, not only has the treatment effect of GLP-1 on diabetes, but also has the treatment effect of GLP-1 on CCK-1, and also has the effect of GLP-1 on CCK-1 on CCK-1. The traditional Chinese medicine composition also has the regulating effect of glucagon on lipolysis and energy metabolism and the beneficial effect of CCK on appetite inhibition, so that a synergistic effect is generated on sugar, fat and energy metabolism; the application has great potential in preparation of drugs for treating metabolic syndromes, such as diabetes, obesity, non-alcoholic fatty liver diseases, dyslipidemia and other diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an incretin analog with multiple biological activities, a synthesis method, a pharmaceutical composition and uses. Background Art

[0002] The dramatic rise in type 2 diabetes mellitus (T2DM) and obesity, known as “diabetic obesity,” is associated with increased mortality and morbidity worldwide. Given this global development, there is a clinical need for antidiabetic therapies that are accompanied by weight loss. Even modest weight loss (approximately 4−5 kg) is an effective means of managing diabetic obesity, with a profoundly beneficial effect on glycemic control and reduced morbidity and mortality. The physiological effects of the gastrointestinal hormone glucagon-like peptide-1 (GLP-1) include improved glycemic control and reduced appetite and food intake, and several GLP-1 receptor agonists are currently on the market. The continued clinical success of GLP-1 receptor agonists reinforces the great potential of this class of drugs for the treatment of both T2DM and obesity, and these agonists remain the only class of drugs approved for both indications. The once-daily injected GLP-1 receptor agonist liraglutide (Saxenda) has been shown to induce average weight loss of 5−10% in nondiabetic patients, and the recently approved once-weekly injected GLP-1 receptor agonist semaglutide (Wegovy) has been shown to induce average weight loss of 15−20% in this population. However, further improvement in weight loss efficacy with GLP-1 receptor agonists alone is limited by dose-dependent gastrointestinal events. Furthermore, at least in patients with diabetes, the maximum weight loss efficacy of GLP-1 receptor agonists does not appear to be further increased with increasing doses.

[0003] Therefore, in order to overcome the limitations of the weight loss effect of GLP-1 receptor agonists, in addition to the currently approved GLP-1 receptor agonists, the next generation of peptide agonists can combine the activity of GLP-1 and other gastrointestinal hormones into one molecule to produce a superior therapeutic effect. Among them, the single molecule co-agonist of the GLP-1 receptor and the glucagon receptor was the first to appear, aiming to utilize the anorexia and anti-diabetic effects of GLP-1, as well as the ability of glucagon to increase energy expenditure, to achieve efficient weight loss. Glucagon, secreted by pancreatic α cells, is an insulin-counterregulatory hormone that increases blood glucose levels by stimulating gluconeogenesis and glycogenolysis, thereby avoiding hypoglycemia. Research data show that in rodents and humans, glucagon may have beneficial effects on energy balance, body fat mass, and nutrient intake.

[0004] There are many endogenous hormones with metabolic benefits in the human body, such as peptide YY and cholecystokinin (CCK), which have certain feeding inhibitory effects. CCK is a peptide hormone in the intestine and brain that plays an important role in the regulation of energy balance and is therefore of potential importance in the context of the increasing global obesity epidemic. The biological effects of CCK are mediated by its two G protein-coupled receptors, namely CCK-1 receptor and CCK-2 receptor. Both CCK-1 receptor and CCK-2 receptor are present in the gastrointestinal tract and central nervous system. CCK mediates appetite regulation through CCK-1 receptor present in gastrointestinal vagal afferent neurons. Other effects mediated by CCK-1 receptor activation include gallbladder contraction, pancreatic enzyme secretion, and delayed gastric emptying. CCK-2 receptor plays a role in anxiety, pain, attention, and memory. The combination of CCK peptide analogs and GLP-1 receptor agonists has shown significant potential for the treatment of T2DM and obesity in rodents. There are currently no anti-obesity drugs targeting the CCK-1 receptor on the market, although there is abundant evidence that CCK or synthetic CCK-1 receptor agonists can reduce food intake in different species including humans, especially when used in combination with other anti-obesity drugs, while CCK-1 receptor antagonists can increase food intake.

[0005] GI181771 is an oral small molecule CCK-1 receptor agonist and CCK-2 receptor antagonist, but failed in clinical trials due to lack of efficacy. GI181771 has low oral bioavailability and low in vitro affinity for the natural CCK-1 receptor ligand CCK-8. CCK peptide analogs may be alternatives to small molecule drugs, but CCK-8 and its longer natural analogs have a half-life of only a few minutes in vivo and have no selective agonist activity for CCK-1 and CCK-2 receptors, with similar potency at CCK receptors. If CCK-8 can improve its in vivo stability and significantly improve its agonist selectivity for CCK-1 receptors to avoid any adverse side effects associated with CCK-2 receptors in central or peripheral locations, CCK-8 may become a new partner to assist GLP-1 receptor agonists in weight loss (J. Med. Chem. 2019, 62, 1407−1419). By combining the beneficial effects of GLP-1 receptor, glucagon receptor and CCK-1 receptor and designing incretin analogs that can act on these three receptors simultaneously, it is possible to simultaneously utilize the beneficial regulatory effects of GLP-1, glucagon and CCK on blood sugar, energy metabolism and food intake, thereby achieving efficient blood sugar lowering and weight loss. Summary of the invention

[0006] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides an incretin analog with multiple biological activities, a synthesis method, a pharmaceutical composition and uses. The incretin analog can act on the GLP-1 receptor, glucagon receptor and CCK-1 receptor simultaneously, and exert the activities of GLP-1, glucagon and CCK simultaneously; it not only has the therapeutic effect of GLP-1 on diabetes, but also has the regulatory effect of glucagon on lipolysis and energy metabolism and the beneficial effect of CCK on appetite suppression, thereby producing a synergistic effect on sugar, fat and energy metabolism; it has great potential for application in the preparation of drugs for the treatment of metabolic syndrome, such as diabetes, obesity, non-alcoholic fatty liver disease and dyslipidemia.

[0007] Technical solution: In the first aspect, the present invention provides an incretin analog with multiple biological activities, wherein the sequence structure of the incretin analog is: .

[0008] In a second aspect, the present invention provides a pharmaceutical composition comprising the incretin analogs described above and a pharmaceutically acceptable carrier, diluent or excipient.

[0009] In a third aspect, the present invention provides a use of an incretin analog as described above or a pharmaceutical composition as described above in the preparation of a medicament for treating a metabolic disease or condition.

[0010] Furthermore, the metabolic disease or condition is diabetes, obesity, non-alcoholic fatty liver disease or dyslipidemia.

[0011] In a fourth aspect, the present invention provides a method for synthesizing the incretin analog as described above, the specific steps are as follows: S1. Swell the Rink Amide-MBHA resin with dichloromethane, and perform Fmoc deprotection and coupling on the swollen resin: take Fmoc-Phe-OH, use HBTU / HOBT / DIPEA to couple the amino acid, and then use 20% piperidine / DMF to remove the Fmoc protecting group; S2. According to the sequence of the peptide chain, repeat the deprotection and coupling steps described in S1 to connect the corresponding amino acids in sequence until the peptide chain is synthesized: for the sulfonated Tyr at position 51, use Fmoc-L-Tyr(SO2(ONeopentyl))-OH; for the Lys site modified by fatty acid, use Fmoc-Lys(Dde)-OH for Lys, and use Boc-His(Trt)-OH for the N-terminal His amino acid; S3. After the peptide backbone is synthesized, Dde deprotection is performed; then, Fmoc-Glu-OtBu, Fmoc-Glu-OtBu and palmitic acid are sequentially added for coupling through the same deprotection and coupling steps as described in S1 to complete the fatty acid modification of the Lys side chain; S4. After the peptide synthesis in S3 is completed, it is cleaved, precipitated, and centrifuged to obtain a crude polypeptide; S5. The sTyr side chain of the crude polypeptide described in S4 is removed from the neopentyl group, followed by purification by reverse phase high performance liquid chromatography to obtain the incretin analog.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following specific beneficial effects: (1) The incretin analogues of the present invention are distinguished from the structure that the amino acid sequence at positions 1-39 on the left is the GLP-1 receptor and glucagon receptor agonist activity region, the amino acid sequence at positions 40-49 (GGGGG GGGGE) is the linker region, and the amino acid sequence at positions 50-57 is the sequence region of CCK-8 (DsYMGW MDF). The compound design method of the present invention using the GLP-1 receptor and glucagon receptor agonist fragments at positions 1-39 on the left and the CCK-8 connected via the amino acid linker fragment at positions 40-49 not only significantly improves the agonist selectivity of CCK-8 for the CCK-1 receptor and avoids the side effects that may be caused by stimulating the CCK-2 receptor, but also achieves the strong GLP-1 receptor and glucagon receptor agonist activity and prolonged in vivo stability of the incretin analogues of the present invention.

[0013] (2) The incretin analogs of the present invention achieve similar in vivo stability to semaglutide through γGlu-γGlu-palmitic acid side chain modification, and can be administered once a week.

[0014] (3) The incretin analogs of the present invention have significantly better blood sugar lowering, weight loss and lipid-regulating effects than semaglutide, and provide new ideas for the design of such drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The percentage of body weight change of the incretin analog provided by the present invention after long-term administration of DIO mice for 21 days; Figure 2 This is a blood glucose curve of a glucose tolerance test conducted on DIO mice after long-term administration of the incretin analog provided by the present invention for 21 days. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the embodiments.

[0017] It should be noted that, unless otherwise defined in the present invention, the scientific and technical terms used in this specification should have the meanings commonly understood by those of ordinary skill in the art.

[0018] Unless explicitly stated otherwise, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0019] In addition to natural amino acids, the sequences of the present invention also use unnatural amino acids, dextrorotatory serine (D-Ser) and sulfotyrosine (sTyr).

[0020] Implementation 1: This embodiment provides an incretin analog with multiple biological activities, SEQ ID NO: 1, whose sequence structure is: .

[0021] The synthesis of the incretin analog SEQ ID NO: 1 is as follows: The peptide chains were constructed by standard Fmoc solid phase synthesis method using Rink Amide-MBHA resin.

[0022] (1) Synthesis of the first amino acid Weigh 0.1 mmol equivalent of Rink Amide-MBHA resin, transfer it to a peptide synthesis tube, and then swell the resin with dichloromethane for half an hour. After the resin is swollen, weigh 0.4 mmol of Fmoc-Phe-OH and add it to the peptide synthesis tube. Use HBTU / HOBT / DIPEA to couple the amino acid for 1-2 hours. Use Kaiser reagent to check whether the reaction coupling is complete. If not, couple it twice. Then, use 20% piperidine / DMF to remove the Fmoc protecting group and proceed to the next coupling.

[0023] (2) Synthesis of polypeptide chains The same coupling method was used to add the corresponding amino acids according to the peptide chain structure for coupling. For the sulfonated Tyr at position 51, neopentyl-protected sTyr (Fmoc-L-Tyr(SO2(ONeopentyl))-OH) was used. For the fatty acid-modified Lys site (position 14), Fmoc-Lys(Dde)-OH was used for Lys, and Boc-His(Trt)-OH was used for the N-terminal His amino acid.

[0024] (3) Modification of fatty acid side chains After the peptide backbone was synthesized, Dde deprotection was performed using 2% hydrazine hydrate / DMF solution. The fatty acid modification of the Lys side chain was constructed using the same Fmoc chemistry as above, with Fmoc-Glu-OtBu, Fmoc-Glu-OtBu and palmitic acid added in sequence for coupling to complete the fatty acid modification of the side chain.

[0025] (4) Peptide cleavage After peptide synthesis was completed, the resin was cleaved with 2.5% water / 2.5% triisopropylsilane / trifluoroacetic acid (TFA) for 2 hours, followed by ether precipitation and centrifugation to obtain the crude peptide.

[0026] (5) Removal of sTyr neopentyl The crude peptide was treated with 2 M ammonium amino acetate in acetonitrile, and the mixture was reacted at 55°C for 12 hours to remove the neopentyl protecting group of sTyr.

[0027] (6) Peptide purification The reaction mixture was purified by reverse phase HPLC preparative chromatography: 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~80% B / 80 min). Methanol was removed by rotary evaporation and pure peptide was collected by lyophilization. Analytical HPLC verified that the purity was greater than or equal to 98%, and MS mass spectrometry confirmed that the molecular weight was the target polypeptide. The theoretical relative molecular mass was 6480.0. ESI-MS m / z: calculated value [M+5H] 5+ 1297.0, [M+6H] 6+ 1081.0; Observed value [M+5H] 5+ 1296.9, [M+6H] 6+ 1080.7. Implementation 2:

[0028] Determination of the agonist activity of incretin analogs on GLP-1 receptor, glucagon receptor and CCK receptor Functional assays of incretin analogs for agonism of GLP-1 and glucagon receptors were performed by measuring the cAMP response of HEK293 cells stably expressing GLP-1 and glucagon 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 glucagon 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.

[0029] HEK293 cells expressing CCK-1 or CCK-2 receptors were added to 384-well plates with 20 µL of coating solution per well, incubated at room temperature for 2 hours, rinsed with 20 µL of sterile water per well, and the target cell solution was added. The medium was removed by aspiration, and the cells were washed with 3 mL of D-PBS and 3 mL of trypsin-EDTA was added. Incubated at 37°C for 1-2 minutes, the cells were peeled off the bottom of the bottle, 3 mL of growth medium was added, and the cells were fully suspended with a pipette and centrifuged at 1000 rpm for 5 minutes. The supernatant was gently poured or aspirated, and the cells were pelleted in 11 mL of medium and 1 mL was removed for cell counting. The cell concentration was counted using ViCell and 20 µL of cell suspension (20,000 cells per well) was added to each well of the 384-well plate. The cells were placed in an incubator at 37°C and 5% CO2 overnight. The test compounds were diluted 10 times by 4 times, and 750 nanoliters of the compounds were transferred to the 384-well compound plate using Echo. Remove media from cell plates and add 20 μL of assay buffer without BSA to each well. Use a pipette to add 20 μL of 2x concentration of Fluo-4 DirectTM to a 384-well cell culture plate without washing loading buffer, for a final volume of 40 μL in the cell plate. Incubate at 37°C, 5% CO2 for 50 minutes and room temperature for 10 minutes. Remove cell plates from incubator and place in FLIPR. Place compound plate and tip box in FLIPR. For agonist plates, run protocol on FLIPRTETRA, transfer 10 μL of compound from DRC plate to cell plate and read fluorescence signal. Determine EC using GraphPad Prism 7.0 50 Numeric value.

[0030]

[0031] As shown in Table 1, the incretin analogs provided by the present invention have strong agonist activity on the GLP-1 receptor, glucagon receptor and CCK-1 receptor. In addition, although the amino acid sequence of positions 50-57 of the incretin analogs of the present invention is the same as that of CCK-8 (DsYMGW MDF), compared with CCK-8, the incretin analogs of the present invention achieve significantly improved CCK-1 receptor selective agonist activity. Implementation 3:

[0032] Pharmacokinetic properties of incretin analogs in rats: SD rats were given a subcutaneous injection of 100 nmol / kg, and blood samples were collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 36 h, and 48 h after administration. Plasma samples were analyzed by LC-MS / MS after protein precipitation with acetonitrile. Pharmacokinetic parameters and half-lives were calculated using WinonLin 5.2.1 (non-compartmental model) (Table 2).

[0033]

[0034] As shown in Table 2, the in vivo half-life of the incretin analogs of the present invention is significantly prolonged, and its in vivo half-life is close to that of semaglutide, which is already on the market and is administered once a week, indicating that the incretin analogs of the present invention have pharmacokinetic characteristics that support once-weekly administration. In addition, it is generally difficult to achieve an in vivo half-life similar to that achieved by 18-alkanediol modification used in the semaglutide side chain by palmitic acid side chain modification (Front Endocrinol (Lausanne), 2019, 10, 155), but the in vivo half-life of the incretin analogs of the present invention is very close to that of semaglutide, achieving an unexpected effect. Implementation 4:

[0035] Effects of incretin analogs on glucose-lowering, lipid-regulating and weight loss in diet-induced obese (DIO) mice Six-week-old male C57BL / 6J mice were fed with a high-fat diet (D12492) from Research Diets for more than 4 months. The DIO mouse model was considered successful when the weight of the mice exceeded 48 grams. Each group of DIO mice was divided into groups according to their weight, and the initial weight of each group of mice was kept similar. There were 6 mice in each group, and the administration cycle was subcutaneous injection every 2 days, including the blank group (normal saline), semaglutide group (30 nmol / kg) and SEQ ID NO: 1 group (30 nmol / kg). The weight of the mice was recorded every other day, and the experimental period was 3 weeks. After the end of the experiment (day 22), the mice in each group fasted for 8 hours, and then an oral glucose tolerance test (OGTT) was performed. Each group of mice was gavaged with 1.5 g / kg of glucose, and then the blood glucose level was measured at 0, 15, 30, 60 and 120 minutes to evaluate the changes in glucose tolerance. On day 23, blood samples were collected from each group of DIO mice in a non-fasting state to prepare serum, and serum triglyceride and cholesterol levels were measured. In addition, the livers of mice were taken out after being sacrificed, and the liver triglyceride content was measured.

[0036]

[0037] like Figure 1 , Figure 2 The results in Table 3 show that the incretin analogs of the present invention can significantly reduce the body weight of DIO mice, which is significantly better than semaglutide that has been on the market, indicating that the reasonable integration of the activities of GLP-1, glucagon and CCK-8, and the design of incretin analogs that can act on GLP-1 receptors, glucagon receptors and CCK-1 receptors can achieve significantly improved weight loss effects. In addition, the incretin analogs of the present invention can also significantly improve the glucose tolerance level of DIO mice, have excellent blood sugar treatment effects, and are significantly better than semaglutide. The above results show that the incretin analogs of the present invention have excellent weight loss and hypoglycemic effects.

[0038]

[0039] As shown in the results of Table 4, the incretin analogs of the present invention can significantly reduce the serum triglyceride and cholesterol levels of DIO mice, and significantly reduce the liver triglyceride content of DIO mice, and have excellent lipid-regulating effects and therapeutic effects on non-alcoholic fatty liver disease. The lipid-regulating effects and therapeutic effects of the incretin analogs of the present invention on non-alcoholic fatty liver disease are also significantly better than semaglutide.

[0040] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

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

3. Use of an incretin analogue as claimed in claim 1 or a pharmaceutical composition as claimed in claim 2 in the preparation of a medicament for treating metabolic diseases or disorders.

4. The use according to claim 3, characterized in that: The metabolic disease or condition is diabetes, obesity, non-alcoholic fatty liver disease or dyslipidemia.

5. A method for synthesizing an incretin analogue according to claim 1, characterized in that: The specific steps are as follows: S1. Swell the Rink Amide-MBHA resin with dichloromethane, and perform Fmoc deprotection and coupling on the swollen resin: take Fmoc-Phe-OH, use HBTU / HOBT / DIPEA to couple the amino acid, and then use 20% piperidine / DMF to remove the Fmoc protecting group; S2. According to the sequence of the peptide chain, repeat the deprotection and coupling steps described in S1 to connect the corresponding amino acids in sequence until the peptide chain is synthesized: for the sulfonated Tyr at position 51, use Fmoc-L-Tyr(SO2(ONeopentyl))-OH; for the Lys site modified by fatty acid, use Fmoc-Lys(Dde)-OH for Lys, and use Boc-His(Trt)-OH for the N-terminal His amino acid; S3. After the peptide backbone is synthesized, Dde deprotection is performed; then, Fmoc-Glu-OtBu, Fmoc-Glu-OtBu and palmitic acid are sequentially added for coupling through the same deprotection and coupling steps as described in S1 to complete the fatty acid modification of the Lys side chain; S4. After the peptide synthesis in S3 is completed, it is cleaved, precipitated, and centrifuged to obtain a crude polypeptide; S5. The sTyr side chain of the crude polypeptide described in S4 is removed from the neopentyl group, followed by purification by reverse phase high performance liquid chromatography to obtain the incretin analog.

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