GLP-1 analogue, preparation method and application in type 2 diabetes mellitus

Abstract

The invention discloses a GLP-1 analogue, a preparation method and application of the GLP-1 analogue in type 2 diabetes. The amino acid sequence of the GLP-1 analogue is as shown in SEQ ID NO. 1; wherein the cysteine at the first position and the cysteine at the sixth position in the sequence form an intramolecular disulfide bond. The polypeptide obtained by substituting, deleting or adding one or more amino acids has the same or similar function with the GLP-1 analogue, or the polypeptide with at least 90% of homology of the amino acid sequence shown in the invention, the specific amino acid sequence and the functional analogue or derivative thereof.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, and in particular to a GLP-1 analogue, a preparation method and an application thereof in type 2 diabetes. Background Art

[0002] Diabetes, especially type 2 diabetes, is a chronic metabolic disease prevalent worldwide with a high prevalence and significant public health burden.

[0003] The pathological mechanism of type 2 diabetes mainly involves insulin resistance and insufficient insulin secretion, which leads to the inability to effectively control the patient's blood sugar level. Long-term high blood sugar can cause a variety of complications, such as cardiovascular disease, kidney disease, neuropathy and retinopathy.

[0004] In recent years, GLP-1 (glucagon-like peptide-1) analogs have received widespread attention as drugs for the treatment of type 2 diabetes. GLP-1 is an incretin hormone that is mainly secreted by L cells in the small intestine. GLP-1 binds to the GLP-1 receptor on pancreatic β cells, promotes insulin secretion, inhibits glucagon release, slows gastric emptying, and suppresses appetite under the action of the central nervous system, thereby effectively lowering blood sugar levels and helping weight management.

[0005] However, natural GLP-1 molecules have a very short half-life in the body (about 1-2 minutes), mainly due to rapid degradation by dipeptidyl peptidase-4 (DPP-4). Therefore, natural GLP-1 is not suitable for use as a drug. In order to overcome this limitation, researchers have developed a variety of GLP-1 analogs, which modify the structure of GLP-1 molecules to improve their stability and bioavailability so that they can continue to function in the body. These GLP-1 analogs include Exenatide, Liraglutide, Dulaglutide, Albiglutide and Semaglutide.

[0006] Although GLP-1 analogs have shown significant therapeutic effects in improving blood sugar control, promoting weight loss, and reducing cardiovascular risks, there are still several limitations and challenges, including gastrointestinal side effects, and as the treatment time increases, some patients may develop tolerance to GLP-1 analogs, resulting in reduced drug efficacy.

[0007] Therefore, developing new GLP-1 analogs to further optimize their efficacy, safety and patient compliance remains an important research direction in this field. In this context, the present invention proposes a new GLP-1 analog, aiming to overcome the limitations of the prior art and provide a more effective and safe treatment solution.

[0008] Although existing GLP-1 analogs have achieved remarkable clinical results in the treatment of type 2 diabetes and obesity, they still have some key shortcomings and limitations:

[0009] Short half-life: Natural GLP-1 and its early analogs have a short half-life in the body and are usually rapidly degraded within a few minutes to an hour. This requires frequent dosing during treatment, which is inconvenient for patients.

[0010] Easy to degrade: GLP-1 analogs are easily degraded by enzymes (such as DPP-4 enzyme) in the body, resulting in rapid loss of their activity.

[0011] Preparation and stability issues: Some GLP-1 analogs may encounter complex process challenges during preparation and are not stable enough during storage and transportation, which increases production costs and management difficulties, limiting the popularity and application of drugs. Summary of the invention

[0012] The purpose of the present invention is to provide a GLP-1 analog, a preparation method and its application in type 2 diabetes. In order to overcome the existing shortcomings of existing GLP-1 analogs, the present invention aims to achieve the following goals: Prolong drug half-life: The present invention provides a GLP-1 analog with enhanced stability, which prolongs its half-life in the body by introducing specific disulfide bonds or chemical modifications in the amino acid sequence, thereby reducing the frequency of administration and improving patient compliance. Enhanced anti-degradation ability: The GLP-1 analog of the present invention is designed to resist DPP-4 enzyme degradation, thereby maintaining activity for a longer period of time, improving the effectiveness of the drug, reducing the dosage, and reducing treatment costs. Improved preparation and stability: By optimizing the production process and molecular design, the GLP-1 analog of the present invention has higher preparation efficiency and stability, and is easier to manage during storage and transportation, thereby reducing production costs and increasing the market competitiveness of the drug.

[0013] A GLP-1 analogue, the amino acid sequence of the GLP-1 analogue is shown in SEQ ID NO.1; wherein the cysteine ​​at position 1 and the cysteine ​​at position 6 in the sequence form an intramolecular disulfide bond.

[0014] As an improvement on a GLP-1 analogue described in the present invention, a polypeptide having the same or similar function as the above-mentioned GLP-1 analogue obtained by replacing, deleting or adding one or more amino acids in the polypeptide, or a polypeptide having at least 90% homology with the amino acid sequence shown, the specific amino acid sequence and its functional analogues or derivatives.

[0015] As a preparation method of a GLP-1 analogue described in the present invention, the steps are as follows: step 1, put 1g of dichloro resin into a 150ml reactor, and add 50ml of dichloromethane to soak for 2 hours; wash the resin with dimethylformamide, and then drain it, repeat this four times, and drain the resin; step 2, weigh 0.3mmol N-[(9H-fluorene-9-ylmethoxy)carbonyl]glycine, 80ml of dichloromethane, and 4ml of N,N-diisopropylethylamine and add them to the reactor, and then place the reactor in a shaking table at 30°C to react for 2 hours; seal it with methanol solution for half an hour, wash it with dimethylformamide four times, and drain it;

[0016] Step 3: Add 20% piperidine solution to the reactor to remove the Fmoc protecting group; wash with dimethylformamide four times and drain; Step 4: weigh 0.9 mmol N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, 60 mg 1-hydroxybenzotriazole, 0.5 ml 1,3-Diisopropylcarbodiimide was added to the reactor, and then the reactor was placed in a shaker at 30°C for 1 hour, the resin was washed four times with dimethylformamide, and then dried; step 5, a certain amount of 20% piperidine was added to the reactor, and it was placed on a decolorizing shaker and shaken for 20 minutes, washed four times with dimethylformamide, and then dried; the remaining amino acids were connected in sequence according to steps 4-5; step 6, a cleavage reagent was used to react for 2 hours to completely remove the polypeptide protecting groups and the resin, and the crude polypeptide was precipitated with ice ether, and the crude polypeptide was dissolved in hydrogen peroxide to form a disulfide bond, and MS was detected to determine whether it was completely oxidized; step 7, the target peptide was separated from the impurities by a high performance liquid chromatography instrument and sent for freeze drying.

[0017] As a further improvement to the method for preparing a GLP-1 analogue described in the present invention, in step three, the ratio of the methanol solution is as follows: methanol: N,N-diisopropylethylamine: dichloromethane = 1:1:2.

[0018] As a further improvement to the method for preparing a GLP-1 analogue described in the present invention, in the step 2, 0.3 mmol N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine is weighed as the first amino acid at the C-terminus.

[0019] As a further improvement to the method for preparing a GLP-1 analogue described in the present invention, in the step 4, 0.9 mmol N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine is weighed as the second amino acid at the C-terminus, and the molar mass is 3 times that of the first amino acid.

[0020] As a further improvement to the method for preparing a GLP-1 analogue described in the present invention, in the step 5, the volume of piperidine added is as follows: piperidine: dimethylformamide = 1:4.

[0021] As a further improvement to the method for preparing a GLP-1 analogue described in the present invention, in step six, the cleavage reagent is a 95% trifluoroacetic acid solution.

[0022] Use of a GLP-1 analog in the treatment of type 2 diabetes.

[0023] The present invention has the following beneficial effects:

[0024] The GLP-1 analogues of the present invention further optimize the amino acid sequence in molecular design, introduce specific intramolecular disulfide bonds or other chemical modifications, and further improve the stability of the molecule through a more stable structure and enhanced anti-degradation modification. In addition, some GLP-1 analogues may encounter complex process challenges during the preparation process and are not stable enough during storage and transportation. The GLP-1 analogues of the present invention have higher preparation efficiency and stability through optimized production process and molecular design, and are easier to manage during storage and transportation, thereby reducing production costs and increasing the market competitiveness of the drug.

[0025] In summary, the present invention provides a more effective, safer and more widely applicable treatment plan for the management and treatment of metabolic diseases such as type 2 diabetes and obesity by designing a new GLP-1 analog. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0027] Figure 1 is the mass spectrum of Example 1 of the present invention;

[0028] Figure 2 This is the analytical liquid phase diagram of Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The technical solution adopted by the present invention is:

[0031] An agonist targeting the GLP-1 receptor, wherein the GLP-1 analog amino acid sequence SEQ-1 is: CHAEGCFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0032] The cysteine ​​at position 1 and cysteine ​​at position 6 in the sequence form an intramolecular disulfide bond. The sequence of the GLP-1 analog is shown below.

[0033]

[0034] Example 1. Synthesis of GLP-1 Analogs

[0035] The weight of each raw material is calculated based on the weight of the target polypeptide (all raw materials are protected amino acids with the corresponding amino acid raw materials at the end).

[0036] Step 1: Place 1 g of dichlororesin into a 150 ml reactor and add 50 ml of dichloromethane (DCM) to soak for 2 hours; wash the resin with dimethylformamide (DMF) and then drain it. Repeat this process four times to drain the resin.

[0037] Step 2: Weigh 0.3 mmol N-[(9H-fluorene-9-ylmethoxy)carbonyl]glycine (Fmoc-Gly-OH) (the first amino acid at the C-terminus) + 80 ml DCM + 4 ml N,N-diisopropylethylamine (DIEA) and add them into the reactor, and then place the reactor in a shaker at 30°C to react for 2 hours.

[0038] Step 3: Block with methanol solution (methanol: DIEA: DCM = 1:1:2) for half an hour, then wash four times with DMF and drain.

[0039] Step 4: Add 20% piperidine solution to the reactor to remove the Fmoc protecting group. After the protection is removed, wash with DMF four times and then drain.

[0040] Step 5: Take a small amount of resin and test it by ninhydrin method. If the resin has color, it means the deprotection is successful.

[0041] Step 6: Weigh 0.9 mmol N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) (the second amino acid at the C-terminus, with a molar mass three times that of the first amino acid) + 60 mg 1-hydroxybenzotriazole (HOBT) + 0.5 ml 1,3-diisopropylcarbodiimide (DIC) and add them into the reactor, and then place the reactor in a shaker at 30°C to react for 1 hour.

[0042] Step 7: Take a small amount of resin for testing using the ninhydrin method. If the resin has color, it means that the condensation is incomplete and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. After the reaction is complete, wash the resin four times with DMF and then drain.

[0043] Step 8: Add a certain amount of 20% piperidine (piperidine / DMF=1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it to check whether the protection is removed.

[0044] Step 9: Take a small amount of resin and test it by ninhydrin method. If the resin has color, it means the deprotection is successful.

[0045] Follow steps 6-9 to connect the remaining amino acids in sequence.

[0046] Step 10: Use a cleavage reagent 95% TFA solution to react for 2 hours to completely remove the polypeptide protective groups and the resin, and use icy ether to precipitate the crude polypeptide.

[0047] Step 11: The crude polypeptide is dissolved in hydrogen peroxide to form disulfide bonds, and MS (mass spectrometry) is detected during the process to determine whether it is completely oxidized.

[0048] Step 12: After complete oxidation, the target peptide is separated from impurities by high performance liquid chromatography (HPLC) and then freeze-dried.

[0049] Step 13: After the target peptide is freeze-dried into powder, the purity is 95.072% after QC inspection. The quality inspection conditions are as follows:

[0050]

[0051] The amino acid raw material table is as follows:

[0052]

[0053]

[0054] Example 2: GLP-1R target agonist cell activity detection experiment

[0055] The HEK293 / CRE-Luc / GLP1R cells in this experiment were purchased from Medicilon Biopharmaceuticals.

[0056] The experimental materials were DMSO, FBS fetal bovine serum, DMEM culture medium, 96-well culture plates, PBS, Bright-Lite Luciferase, Trypsin-EDTA (0.25%), G418 Sulfate, and Hygromycin B. DMSO was purchased from Sigma with the product number D4540; FBS fetal bovine serum was purchased from Gibco with the product number 10099-141; DMEM culture medium was purchased from BasalMedia with the product number L110KJ; 96-well culture plates were purchased from Corning with the product number 3610; PBS was purchased from Pricella with the product number PB180327; Bright-Lite Luciferase was purchased from Vazyme with the product number DD1204-03; Trypsin-EDTA (0.25%) was purchased from Yishen with the product number 40127ES60; Sulfate is purchased from Invivogen brand with the item number Ant-hg-1. Hygromycin B is purchased from Invivogen brand with the item number Ant-hg.

[0057] The experimental instruments include desktop microplate reader, inverted microscope, carbon dioxide incubator, microplate constant temperature oscillation, automatic cell counter, and low-speed multi-tube rack automatic. The desktop microplate reader is from Molecular Devices, model SpectraMax M5; the inverted microscope is from OLYMPUS, model IX71; the carbon dioxide incubator is from Thermo, model 3111; the microplate constant temperature oscillation is from Haimen Qilin Bell Instrument Manufacturing Co., Ltd., model MH-2; the automatic cell counter is from Cellomerer, model AutoT4; the low-speed multi-tube rack automatic is from Hunan Saite Xiangyi Centrifuge Instrument Co., Ltd., model L-530.

[0058] The experimental method is as follows:

[0059] Preparation of test medium and diluent

[0060] Prepare plating culture medium and store it at 2-8℃, the shelf life is 6 months.

[0061] Preparation method: Mix 90% DMEM and 5% FBS (for example: DMEM 450ml, FBS 50ml).

[0062] Prepare the subculture medium: DMEM, 10% FBS, 300 μg / ml G418, 50 μg / ml Hygromycin B and mix well.

[0063] Prepare the detection solution. Use the Bright-Lite Luciferase Assay System to add 100 ml of staining solution to the substrate and mix well to obtain the detection solution.

[0064] To prepare sample gradient solution, a certain amount of sample and reference standard were weighed and dissolved with sterile water to prepare a 1 mM stock solution, followed by the preparation of gradient dilution solution.

[0065] For cell inoculation, the cells were passaged and amplified in advance. When the cell density reached 80%, the cells were washed 1-2 times with PBS, and the cells were digested with 0.25% trypsin. The digestion was terminated after the cells became round under microscope observation. The cells with viability greater than 95% were taken for trypan blue staining and prepared into a cell suspension with a density of 6x105 cells / ml. 100μl of the cell suspension was inoculated into each well of a 96-well plate.

[0066] After drug treatment, 20 μl of serially diluted samples were added to each well the next day and the cells were transferred to a 37°C cell culture incubator and incubated for 3 h.

[0067] After the incubation, the cell plate was removed and placed at room temperature for 15 minutes to allow it to equilibrate to room temperature. Then, the test solution equilibrated to room temperature was added, 80 μl per well. The culture plate was placed on a microplate constant temperature oscillator and shaken at 500 rpm for 3 minutes. Finally, the data was collected using an ELISA reader.

[0068] The experimental data corresponding to the compounds are shown in the following table:

[0069] sample EC50(pM) Semaglutide 127.7 SEQ-1 376.8

[0070] It can be seen from the data in the above table that the GLP-1 analogs of the present invention exhibit the ability to activate the GLP-1 receptor.

[0071] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0072] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A GLP-1 analogue, characterized in that: The amino acid sequence of the GLP-1 analog is shown in SEQ ID NO.1; The first cysteine ​​and the sixth cysteine ​​in the sequence form an intramolecular disulfide bond.

2. A GLP-1 analogue according to claim 1, characterized in that: A polypeptide having the same or similar function as the above-mentioned GLP-1 analogue obtained by replacing, deleting or adding one or more amino acids in the polypeptide, or a polypeptide having at least 90% homology with the amino acid sequence shown, the specific amino acid sequence and its functional analogues or derivatives.

3. A method for preparing a GLP-1 analogue, characterized in that: Here are the steps: Step 1: Place 1g of dichlororesin into a 150ml reactor and add 50ml of dichloromethane to soak for 2 hours; wash the resin with dimethylformamide and then drain it, repeat this four times to drain the resin; Step 2: Weigh 0.3 mmol N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine, 80 ml dichloromethane, and 4 ml N,N-diisopropylethylamine into a reactor, and then place the reactor in a shaker at 30°C for 2 hours; seal with methanol solution for half an hour, wash four times with dimethylformamide, and drain; Step 3: Add 20% piperidine solution to the reactor to remove the Fmoc protecting group; wash with dimethylformamide four times and drain; Step 4: Weigh 0.9 mmol N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, 60 mg 1-hydroxybenzotriazole, and 0.5 ml 1,3-diisopropylcarbodiimide into a reactor, and then place the reactor in a shaker at 30° C. to react for 1 hour, wash the resin four times with dimethylformamide, and then drain; Step 5: Add a certain amount of 20% piperidine to the reactor, shake it on a decolorizing shaker for 20 minutes, wash it four times with dimethylformamide, and then drain it; Connect the remaining amino acids in sequence according to steps 4-5; Step 6: Use a cleavage reagent to react for 2 hours to remove all the polypeptide protective groups and resin, and use ice ether to precipitate the crude polypeptide. The crude polypeptide is dissolved in hydrogen peroxide to form a disulfide bond, and MS is detected to determine whether it is completely oxidized; Step 7: Separate the target peptide from impurities using a high performance liquid chromatography instrument and send for freeze-drying.

4. The method for preparing a targeted GLP-1 analogue according to claim 3, characterized in that: In the step 3, the ratio of the methanol solution is as follows: Methanol: N,N-diisopropylethylamine: dichloromethane = 1:1:

2.

5. The method for preparing a targeted GLP-1 analogue according to claim 3, characterized in that: In the step 2, 0.3 mmol of N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine is weighed as the first amino acid at the C-terminus.

6. The method for preparing a targeted GLP-1 analogue according to claim 3, characterized in that: In the step 4, 0.9 mmol of N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine is weighed as the second amino acid at the C-terminus, and the molar mass is 3 times that of the first amino acid.

7. The method for preparing a targeted GLP-1 analogue according to claim 3, characterized in that: In the step 5, the capacity of adding piperidine is as follows: Piperidine:dimethylformamide=1:

4.

8. The method for preparing a targeted GLP-1 analogue according to claim 3, characterized in that: In the step six, the cutting reagent is 95% trifluoroacetic acid solution.

9. Use of a GLP-1 analogue in the treatment of type 2 diabetes.

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