CN105753963B - Highly active exenatide analogs and their medical applications - Google Patents

Abstract

The invention discloses a kind of Exenatide (Exendin-4) analogs.The activity of such analog stimulation rat Islet cells oncocyte INS-1 excreting insulin is apparently higher than wild type Exenatide, and hypoglycemic activity is also significantly better than wild type Exenatide in diabetic mice C57BL/KsJ-db/db body, can be used for preventing, treating or mitigating diabetes and obesity and the other diseases by reducing blood glucose, inhibiting gastrointestinal motility, gastric emptying benefited.

Description

Highly active exenatide analogs and their medical applications

technical field

The invention belongs to the field of biomedicine, and specifically relates to a class of exendin-4 analogues and medical applications.

technical background

Diabetes mellitus (DM) is a complex metabolic disease in which blood sugar rises due to endocrine and metabolic disorders caused by insufficient insulin secretion or synthesis blockage in the body. It is mainly divided into type 1 diabetes mellitus (T1DM) and type 2 diabetes. (type2diabetes mellitus, T2DM) and other types of diabetes. According to statistics, type 2 diabetes accounts for a large proportion, accounting for more than 90% of the total number of diabetic patients. Its onset period is mostly after the age of 35-40, and more serious complications will appear in the later stage of the onset. At present, the treatment of diabetes mainly focuses on lowering blood sugar concentration and reducing the occurrence of complications, etc., and the treatment method is mainly drug therapy. The drugs for the treatment of type 2 diabetes mainly include sulfonylureas, biguanides, α-glucokinase inhibitors, anti-IL-1β antibodies and incretin drugs. Therapeutic drugs have attracted much attention, and can be divided into dipeptidyl peptidase IV (dipeptidylpeptidase 4, DPP-4) inhibitors and glucagon-like peptide (glucagon-like peptide-1, GLP-1) receptor agonists according to the mechanism of action two types of agents. Studies have shown that these two types of drugs not only have the advantages of lowering blood sugar, rarely causing hypoglycemia, good safety and tolerance, but also exert protective effects on multiple systems such as digestion, central nervous system, and cardiovascular system.

Glucagon-like peptide-1 (GLP-1) is synthesized and secreted by Langerhans cells in the small intestine. After binding to the GLP-1 receptor, it can promote the biosynthesis of insulin with the increase of blood sugar, stimulate Pancreatic beta cells secrete insulin, inhibit the secretion of glucagon, enhance tissue sensitivity to insulin, thereby reducing blood sugar concentration, and have a good therapeutic effect on diabetes. However, GLP-1 is easily degraded by dipeptidyl peptidase IV (dipeptidylpeptidase 4, DPP-4) after being secreted into the blood, and its half-life is only 1-2min. DPP-4 is a serine protease that specifically cleaves the N-terminal dipeptide of GLP-1 to inactivate it.

Exendin-4 is a GLP-1 receptor agonist isolated from the saliva of Heloderma suspectum in northwestern North America, consisting of 39 amino acids (amino acid sequence as SEQ.ID NO: 1), has about 53% homology with GLP-1 amino acid sequence, and its N-terminal homology reaches 80%. Studies have found that exenatide, similar to GLP-1, can also bind and activate GLP-1 receptors on pancreatic β cells, and stimulate glucose-dependent insulin secretion. Exenatide was developed and marketed by American companies Amylin and Eli Lilly in April 2005, and was launched in China in August 2009. It is clinically used in combination with sulfonylureas, metformin or thiazolidinediones. Blood glucose concentration in patients with type 2 diabetes has become the first-line treatment for type 2 diabetes. Exenatide has good safety and tolerance in vivo, and rarely causes hypoglycemia. In addition, because exenatide is not sensitive to dipeptidyl peptidase IV, compared with GLP-1, its half-life in vivo is significantly increased, reaching 3.3 to 4 hours (Drugs Today (Barc) 41 (2005) 563-578.; Am J Health Syst Pharm 62 (2005) 173-181.).

Exenatide is a heterologous GLP-1 receptor agonist, and its structure and interaction with GLP-1 receptors have been studied relatively clearly (Br J Pharmacol 166(2012) 27-41). Therefore, based on the interaction between exenatide and GLP-1 receptors, it is possible to design and obtain GPL-1 receptor agonist drugs with better pharmacological activity.

Contents of the invention

The present invention is based on the structural relationship between exenatide and the GLP-1 receptor of pancreatic beta cells, and according to the principle of computer-aided drug design, by mutating the key sites of exenatide, the GLP-1 receptor Exenatide analogs with stronger binding and higher insulin secretion-stimulating activity.

Concrete technical scheme of the present invention is as follows:

A class of exenatide analogues, the amino acids at one or more positions in the 16th, 21st, and 29th positions of the wild-type exenatide (Exendin-4, EX-WT) amino acid sequence are mutated, wherein, The Glu mutation at the 16th position of exenatide is Trp, Leu, Ile or Val; the Leu mutation at the 21st position of exenatide is Arg, Lys or His; the Gly mutation at the 29th position of exenatide is Trp, Leu, Ile or Phe.

Exendin-4 is a GLP-1 receptor agonist isolated from the saliva of Heloderma suspectum in northwestern North America. It consists of 39 amino acids, and its amino acid sequence is shown below.

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ. ID NO: 1).

Further, the preferred amino acid sequence of the exenatide analogue is shown in SEQ.ID NO: 2-15.

Among them, the 16th Glu of exenatide is mutated into an exenatide analogue of Trp, Ile, Leu or Val, and its amino acid sequence is as shown in SEQ.ID NO:2, SEQ.ID NO:3, SEQ.ID NO: 4, as shown in SEQ.ID NO:5;

The amino acid sequence of exenatide analogues in which Leu at position 21 of exenatide is mutated to Arg, Lys or His is shown in SEQ.ID NO:6, SEQ.ID NO:7, SEQ.ID NO:8;

Exenatide 29th position Gly is mutated to Trp, Ile, Leu or Phe. The amino acid sequence of exenatide analogs is as shown in SEQ.ID NO:9, SEQ.ID NO:10, SEQ.ID NO:11, SEQ.ID NO:11, SEQ. .ID NO: 12;

The 16th Glu of Exenatide is mutated to Trp and the 21st Leu is mutated to Arg or Lys. The two double mutants are EX-E16WL21K (SEQ.ID NO:13) and EX-E16WL21R (SEQ.ID NO: 14)

The 16th Glu of exenatide was mutated to Trp, and the 29th Gly was mutated to Trp, resulting in a double mutant EX-E16WG29W (SEQ. ID NO: 15).

The C-terminus of wild-type exenatide is amidated, and the amino acid C-terminus of the exenatide analogs of the present invention can be amidated or non-amidated.

Another object of the present invention is to provide the application of the exenatide analogs of the present invention in the preparation of drugs for treating diabetes, obesity and/or related diseases.

For the above application, the drug contains the exenatide analogue of the present invention and a pharmaceutically acceptable carrier.

The exenatide analogs of the present invention can be administered as a single drug or in combination with other drugs.

Exenatide analogs of the present invention can be salted, including various inorganic or organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, horse Tonate, fumarate, mandelate and oxalate; various inorganic or organic bases, salts such as sodium hydroxide, trishydroxymethylaminomethane and N-methyl-glucosamine into salt.

The exenatide analogues of the present invention can be used alone or in the form of pharmaceutical compositions. The pharmaceutical composition comprises the exenatide analogue of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" will not destroy the pharmaceutical activity of the compound of the present invention or its pharmaceutically acceptable salt, and at the same time, its effective dosage, that is, the dosage when it can function as a pharmaceutical carrier, is non-toxic to human body. "Pharmaceutically acceptable carriers" include, but are not limited to: ion exchange materials, aluminum oxide, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-vitamin E polyethylene glycol 1000 succinate, Surfactants for pharmaceutical preparations such as Tween or other similar polymerization media, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, a mixture of saturated vegetable fatty acid partial glycerides, water, Salt, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, silica gel, magnesium silicate, etc. Polyvinylpyrrolidone, cellulosic substances, polyvinyl alcohol, sodium carboxymethylcellulose, polyacrylates, ethylene-polyoxyethylene-block polymers and lanolin, cyclodextrins such as α-, β-, γ- Cyclodextrins or their chemically modified derivatives such as hydroxyalkylcyclodextrins such as 2- and 3-hydroxypropyl-β-cyclodextrins or other soluble derivatives can be used to facilitate the compounds of the present invention, their pharmaceutical Drug delivery using salts or prodrugs.

Other pharmaceutically acceptable excipients such as fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as cross-linked sodium carboxymethyl starch, cross-linked carboxymethyl starch Sodium methylcellulose, low-substituted hydroxypropylcellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents , solvents, solubilizers and colorants, etc. can also be added to the pharmaceutical composition of the present invention.

The above-mentioned exenatide analogs or their pharmaceutically acceptable salts and pharmaceutical compositions of the present invention can be administered through enteral or parenteral routes. Preparations for parenteral administration include injections, creams, ointments, patches, sprays and the like. Routes of administration include subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, or, oral, topical, rectal, trans Nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethral administration, or administration by inhalation of aerosol or implant accumulation or acupuncture.

The Exenatide analogue or its pharmaceutically acceptable salt and the pharmaceutical composition of the present invention can be used for single drug or combined drug treatment of related diseases, which is within the scope understood by those skilled in the art.

The results of pharmacodynamic experiments prove that the activity of exenatide analogues disclosed in the present invention in stimulating rat islet cell tumor cells INS-1 to secrete insulin is significantly higher than that of wild-type exenatide, and the diabetic model mice C57BL/KsJ -db/db (ie, BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice) in vivo hypoglycemic activity was also significantly better than wild-type exenatide. Therefore, the analogs can be used to better prevent, treat or alleviate diabetes and obesity and other diseases that benefit by lowering blood sugar, inhibiting gastrointestinal motility, and gastric emptying.

Use MOE software (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada) to analyze the difference between exenatide analogs of the present invention and wild-type exenatide, the results show that compared with wild-type, exenatide is similar The number of interactions between the drug and the receptor increases, and exenatide at the mutation sites 16 and 19 forms a new hydrophobic interaction with the receptor, thereby increasing the binding energy with the receptor and improving the activity. . Exenatide 21-site mutations form a new intra-chain ionic bond with Glu17, which improves the stability of the α-helix of exenatide, thereby increasing the activity.

Description of drawings

Figure 1: Effects of exenatide and its analogues on insulin secretion activity (GSIS/BIS value) of rat insulinoma INS-1 cells (n=3, means±SEM). ^# , ^## and ^#### represent p<0.05, p<0.01 and p<0.0001 compared with normal control group; ^* , ^*** and ^**** represent p<0.05,p compared with EX-WT group <0.001 and p<0.0001. The experiment was repeated three times.

Figure 2: Effects of exenatide and its analogues on blood glucose levels in BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice after intraperitoneal administration at a dose of 10 nmol/kg (n=10, Means±SEM). ^#### indicates p<0.0001 compared with the normal group; ^*** and ^**** indicate p<0.001 and p<0.0001 compared with the model group.

Figure 3: Effects of exenatide and its analogs on blood glucose levels in BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice after intraperitoneal administration at a dose of 10 nmol/kg (n=10, AUC _0-24h ) . ^#### indicates p<0.0001 compared with the normal group; ^*** and ^**** indicate p<0.001 and p<0.0001 compared with the model group; ^Δ and ^ΔΔ indicate p<0.05 and p<0.05 compared with the EX-WT group 0.01; ns means p>0.05 compared with EX-WT group.

Figure 4: Effects of exenatide and its analogs on intraperitoneal glucose tolerance IPGTT in BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice (n=10, means±SEM).

Fig. 5: Effects of exenatide and its analogues on IPGTT of intraperitoneal glucose tolerance in diabetic mice (n=10, AUC _0-180min ). ^#### indicates p<0.0001 compared with the normal group; ^**** indicates p<0.0001 compared with the model group.

Figure 6: Schematic diagram of the interaction between the 16th amino acid Glu of wild-type Exenatide EX-WT and the receptor.

Figure 7: Schematic diagram of the interaction between the 29th Gly of wild-type exenatide EX-WT and the receptor.

Figure 8: Trp16 in mutant EX-E16W forms two new hydrophobic interactions with Val30 and Trp91 in the receptor, and forms a new hydrogen bond with Glu128 in the receptor.

Figure 9: Trp29 in mutant EX-G29W forms a new hydrophobic interaction with Leu118 in the receptor.

Figure 10: The 21st Leu in the wild-type Exenatide EX-WT does not interact with the 17th Glu.

Figure 11: A new intrachain ionic bond is formed between Lys21 and its 17th Glu in mutant EX-L21K.

Detailed ways

The present invention is further illustrated by the following examples, but this is not meant to limit the scope of the present invention.

Example 1 Design of Exenatide Analogs

Design 1: Taking the binding energy of exenatide analogs and GLP-1 receptor as the investigation index, the crystal structure (PDB ID: 3C5T) (J Biol Chem.283( 2008) 11340–11347) as the object, using FoldX software (J Mol Biol.320 (2002) 369–387; Proc Natl Acad Sci US A.102 (2005) 10147–10152) to design, and obtain several preparations with improved binding energy. Selected mutants include EX-E16W (SEQ ID No: 2), EX-R20M (SEQ ID No: 16), EX-K27M (SEQ ID No: 17) and EX-G29W (SEQ ID No: 9).

Design 2: Because the stability of the α-helical region in the middle of the exenatide molecule is positively correlated with its activity (Biochemistry.46(2007)5830-5840). The design idea is to introduce more intra-chain ionic bonds into the α-helix of exenatide to improve its activity. After analyzing the three-dimensional structure of exenatide, the inventors found that the glutamic acid at position 17 and Leu21 are distributed in the two adjacent helical loops of the α-helix. A new intra-chain ionic bond to obtain a more active exenatide analogue: EX-L21K (SEQ ID No: 7).

Design 3: Combine the single mutation methods of Design 1 and 2 to design double mutants EX-E16WR20M (SEQ ID No: 18), EX-E16WL21K (SEQ ID No: 13), EX-E16WK27M (SEQ ID No: 19) and EX-E16WG29W (SEQ ID No: 15).

Nanjing Jinsirui Biotechnology Co., Ltd. was entrusted to fully synthesize the exenatide analogues designed by the above-mentioned methods 1, 2, and 3.

Example 2 Exenatide analogs in vitro stimulation of rat islet cell tumor cells INS-1 secretion of insulin activity screening

Rat islet cell tumor cells INS-1 were cultured in RPMI-1640 medium until the confluency was about 80%, the original solution was discarded, washed twice with PBS buffer, digested with 0.05% trypsin at 37°C for 2min, and washed with RPMI-10% FBS Stop digestion at 1640, centrifuge at 800rpm for 5min to collect cells, resuspend in cell culture medium and inoculate ⁵ ×105 cells/well into 12-well cell culture plate, culture in 37°C, 5% _CO2 incubator for 24h, discard the original solution , washed once with PBS for the experiment. Experimental grouping: 1, basal insulin secretion group (containing 0, 30, 60nM) exenatide and the serum-free medium of similar drugs designed in Example 1 of the present invention were treated for 24 hours, and each drug concentration was established with 3 duplicate holes) ; 2. Glucose-stimulated insulin secretion group (serum-free medium containing 0, 30, 60 nM exenatide or similar drugs was treated for 24 hours, and 3 replicate wells were set for each drug concentration). The cells of each group were first treated with the medium containing or not containing drugs for 24 hours, and then 200 μl of KRBH buffer containing 3 mM glucose was added to each well of the cells to equilibrate for 1 hour. After discarding the stock solution, add 200 μl of KRBH buffer containing 3 mM glucose to each well of the basal insulin secretion group; add 200 μl of KRBH buffer containing 20 mM glucose to each well of the glucose-stimulated insulin secretion group. After acting at 37°C for 2 hours, the supernatant was collected, and the amount of insulin secretion was detected with a rat hypersensitive insulin ELISA detection kit (Mercodia, catalog number 10-1251-01). At the same time, 200 μl of cell lysate was added to each well, lysed on ice for 30 min, total protein was extracted, and the total protein content of each well was determined by BCA method. Calculate the BIS value of the basal insulin secretion group and the GSIS value of the glucose-stimulated insulin secretion group respectively according to the following formulas.

The screening results showed that among the numerous exenatide analogs designed in Example 1, EX-E16W (SEQ ID No: 2), EX-L21K (SEQ ID No: 7), EX-G29W (SEQ ID No: 9) and EX-E16WG29W (SEQ ID No: 15), which have stronger activity. As shown in Figure 1, under the intervention of different drug concentrations, the biological activity (GSIS/BIS value) of wild-type exenatide and its analogs to stimulate insulin secretion from rat islet cell tumor cells INS-1 increased in a dose-dependent manner. Compared with the normal control group, the low concentration of EX-E16W can significantly promote the insulin secretion of INS-1 cells (p<0.01), and the effect of high concentration is more significant (p<0.0001); compared with the normal control group, EX-L21K and EX- Low concentration of E16WG29W has a very significant promoting effect (p<0.0001); compared with the normal control group, wild-type exenatide EX-WT and EX-G29W at high concentrations can significantly promote insulin secretion (p<0.05 and p<0.05). 0.01). Compared with wild-type exenatide EX-WT, EX-E16W, EX-L21K and EX-E16WG29W had obvious advantages at low concentrations (p<0.05, p<0.0001 and p<0.0001). It can be seen that exenatide and its analogs can promote insulin secretion in INS-1 cells in a dose-dependent manner, and exenatide analogs EX-E16W, EX-L21K and EX-E16WG29W have stronger activities and promote insulin secretion. The secretion effect was significantly better than that of wild-type exenatide.

Example 3 Type II diabetes model mouse C57BL/KsJ-db/db hypoglycemic test in vivo

Six-week-old type II diabetes model mice C57BL/KsJ-db/db (i.e. BKS.Cg-Dock7 ^m +/+ Lepr ^db /JNju mice) male 50 and control mice C57BLKS/JNju 10 were purchased from Nanjing University— Nanjing Institute of Biomedicine (certificate number: 201600019, license number: SCXK (Su) 2015-0001). The mice were kept in an SPF grade animal room with a room temperature of 25° C., a humidity of 40-60%, and 12 hours of light and dark. The experiment was carried out after one week of adaptive feeding. Blood glucose concentration using Roche Performa blood glucose meter and blood glucose test strips for testing. The experimental groups and administration methods are shown in Table 1.

Table 1 Grouping and administration of experimental animals

group mouse no drug Dosage Dosing volume Method of administration 1 C57BLKS/JNju 10 NS normal saline 0.01ml/g ip. 2 BKS.Cg-Dock7^m+/+Lepr^db/JNju 10 NS normal saline 0.01ml/g ip. 3 BKS.Cg-Dock7^m+/+Lepr^db/JNju 10 EX-WT 10nmol/kg 0.01ml/g ip. 4 BKS.Cg-Dock7^m+/+Lepr^db/JNju 10 EX-E16W 10nmol/kg 0.01ml/g ip. 5 BKS.Cg-Dock7^m+/+Lepr^db/JNju 10 EX-L21K 10nmol/kg 0.01ml/g ip. 6 BKS.Cg-Dock7^m+/+Lepr^db/JNju 10 EX-G29W 10nmol/kg 0.01ml/g ip.

1. Acute hypoglycemic test

Before the administration, the blood glucose levels of the mice in each group were detected (see Table 2, Figure 2). The results showed that before administration (0 hour), the blood glucose concentrations in the model group, EX-WT, EX-E16W, EX-G29W, and EX-L21K groups were 22.64±1.60, 23.10±1.44, 22.64±1.48, 22.51±1.45, 22.28±1.51mM, compared with the normal control group, the difference was extremely significant (p<0.0001), but there was no difference between the model group and each administration group, indicating that C57BL/KsJ-db/db (ie BKS.Cg-Dock7 ^m + /+Lepr ^db /JNju) mouse type II diabetes model has been established, and there is no difference between the drug group and the model group, and subsequent drug efficacy experiments can be carried out.

After intraperitoneal administration at a dose of 10 nmol/kg, blood glucose levels were continuously monitored 1, 2, 4, 8, 12, and 24 hours after administration (Table 2, Figure 2). The results showed that after 1 hour of administration, the blood glucose in the EX-WT, EX-E16W, EX-G29W, and EX-L21K groups dropped to 9.36±1.02, 7.48±0.67, 5.70±0.34, 7.66±0.84mM, compared with the model group Significantly (p<0.0001). After 8 hours of administration, each administration group still maintained a very low blood glucose level (p<0.0001vs model group). After 24 hours of administration, the blood glucose in the EX-WT group had risen to 21.33±1.51mM, while the blood glucose in the EX-E16W and EX-L21K groups remained at 15.09±1.69 and 14.27±1.26mM. Compared with the model group, the blood glucose concentration was significantly different ( p<0.001 and p<0.0001), indicating that its hypoglycemic effect is better than that of EX-WT.

Table 2 Effects of exenatide and its analogs on blood glucose levels in BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice after intraperitoneal administration (n=10, Means±SEM)

Note: ^#### indicates p<0.0001 compared with the normal group; ^*** and ^**** indicate p<0.001 and p<0.0001 compared with the model group.

The hypoglycemic effect of exenatide and its mutants was further evaluated by analyzing the area under the curve (AUC) method, and the results are shown in Table 3 and Figure 3. Compared with the normal control group, the AUC _0-24h of the model group increased significantly (p<0.0001). After intraperitoneal injection at a dose of 10nmol/kg, the AUC of EX-WT, EX-E16W, EX-G29W, and EX-L21K groups decreased to 24273±1822mM min (p<0.001vsmodel group) and 18653± _1622mM respectively at 0-24h ·min (p<0.0001 vsmodel group), 21365±1047mM·min(p<0.001vsmodel group), 16958±877mM·min(p<0.001vsmodel group). Compared with the wild-type exenatide EX-WT, the AUC _0-24h of the mutants EX-E16W (p<0.05) and EX-L21K (p<0.01) decreased more significantly. This shows that the four drugs EX-WT, EX-E16W, EX-G29W and EX-L21K all have hypoglycemic effects, especially EX-E16W and EX-L21K have more significant hypoglycemic activities.

Table 3 Effects of exenatide and its analogs on blood glucose levels in BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice after intraperitoneal injection (n=10, AUC _0-24h )

group AUC_0-24h(mM·min) CON 11370±226.8 MOD 32339±1512^#### EX-WT 24273±1822^*** EX-E16W 18653±1622^****Δ EX-G29W 21365±1047^**** EX-L21K 16958±877^****ΔΔ

Note: ^#### p<0.0001vs.control group; ^*** p<0.001and ^**** p<0.0001vsmodel group; ^Δ p<0.05, ^ΔΔ p<0.01and ns p>0.05vs EX-WT group .

2. Intraperitoneal Glucose Tolerance Test (IPGTT)

After the mice were fasted overnight for 12 hours, they were intraperitoneally injected with normal saline or administered at a dose of 10 nmol/kg. After 2 hours of administration, each mouse was injected with 1.5 g/kg glucose intraperitoneally, and blood was taken from the tail vein at 0, 15, 30, 45, 60, 90, 120, and 180 minutes to determine the blood glucose level. During the experiment, the animals were given normal diet and water supply.

Table 4 Effects of exenatide and its analogs on intraperitoneal glucose tolerance IPGTT in BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju mice (n=10, means±SEM)

The results showed that after intraperitoneal injection of 1.5g/kg glucose for 15 minutes, the blood glucose of the mice in the model group rose from 14.46±0.90 to 25.43±2.12mM, and remained at a high level until 180 minutes after the administration of glucose, it dropped to the pre-sugar level. However, after EX-WT, EX-E16W, EX-G29W and EX-L21K 4 were injected intraperitoneally with glucose for 15 minutes, the blood glucose was only increased by 7.64±0.44, 7.65±0.79, 6.16±0.44, 7.02±0.67mM respectively. to 13.03±1.47, 12.84±1.33, 11.64±0.89, 14.87±1.36mM, and remained at a low level until 180 minutes after sugar administration, it did not rise to the level before sugar administration (Table 4 and Figure 4).

The method of area under the curve (AUC) was further analyzed. The results showed that compared with the normal control group, the AUC _0-180min of the model group increased significantly (p<0.0001). In the drug group, after 120 minutes of administration of EX-WT, EX-E16W, EX-G29W and EX- _L21K , 1.5 g/kg of glucose was injected intraperitoneally, and the AUC decreased to 1365±146.0, 1599±184.0, 1144±105.9 respectively in 0-180 minutes , 1667±147.4mM·min. It can be seen that exenatide and its analogs can significantly enhance the tolerance of diabetic mice to glucose (Table 5, Figure 5).

Table 5 Effects of exenatide and its analogs on exenatide and its mutants on the IPGTT of the peritoneal glucose tolerance of diabetic mice (n=10, AUC _0-180min )

group AUC_0-180(mM·min) CON 1479±83.85 MOD 4017±247.5^#### EX-WT 1365±146.0^**** EX-E16W 1599±184.0^**** EX-G29W 1144±105.9^**** EX-L21K 1667±147.4^****

Note: ^#### indicates p<0.0001 compared with the normal group; ^**** indicates p<0.0001 compared with the model group.

Example 4

Differences between the interaction of mutants EX-E16W, EX-G29W and wild-type exenatide EX-WT with the GLP-1 receptor were analyzed using MOE software (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada).

The results showed that, compared with the wild type, the number of interactions between the mutants EX-E16W and EX-G29W increased, that is, new interactions were formed between the mutant sites and the receptor.

Fig. 6 is a schematic diagram of the interaction between the 16th Glu of wild-type exenatide EX-WT and the receptor. Fig. 7 is a schematic diagram of the interaction between the 29th Gly of wild-type exenatide EX-WT and the receptor. Figure 6 and Figure 7 show that there is no interaction between the 16th Glu and the 29th Gly of wild-type exenatide EX-WT and the GLP-1 receptor.

However, in the mutant EX-E16W, the mutated Trp16 forms two new hydrophobic interactions with Val30 and Trp91 in the receptor, and forms a new hydrogen bond with Glu128 in the receptor (as shown in Figure 8).

In mutant EX-G29W, the mutated Trp29 forms a new hydrophobic interaction with Leu118 in the receptor (as shown in FIG. 9 ).

In mutant EX-E16W and mutant EX-G29W, the newly formed interaction at the mutation site is dominated by hydrophobic interaction, so if other hydrophobic amino acids are used to replace the original amino acids at the 16th and 29th positions, Theoretically, it should also be able to form a new hydrophobic interaction with the receptor, thereby increasing the binding energy between it and the receptor, and obtain an activity enhancement effect similar to that of the mutants EX-E16W and EX-G29W. Considering the influence of amino acid side chain length, at the 16th position, replacing Trp in the mutant with Ile, Leu or Val can produce similar activity-enhancing effects; while at the 29th position, replacing the mutant with Ile, Leu, or Phe Body Trp can also produce a similar effect.

Analysis using MOE software showed that the 21st Leu in the wild-type exenatide did not form an interaction with its 17th Glu (Figure 10), while in the mutant EX-L21K, a new interaction was formed between the mutated Lys21 and its Glu17 Intra-chain ionic bonds are revealed (Figure 11). The experimental results confirm the technical idea of the design 2 of the embodiment 1. Since Arg, His and Lys are both basic amino acids (positively charged), they can theoretically form ionic bonds with the 17th acidic amino acid Glu (negatively charged), so Arg and His were used to replace the mutant EX-L21K. The Lys at the 21st position can also form a new intra-chain ionic bond with the Glu at the 17th position, thereby improving the stability of the α-helix of exenatide, and obtaining an activity-enhancing effect similar to that of the mutant EX-L21K.

Claims (4)

1. An exenatide analogue, characterized in that the amino acid sequence is as shown in SEQ. ID NO: 15. 2. The exenatide analogue according to claim 1, characterized in that the amino acid C-terminus of the exenatide analogue is amidated or non-amidated. 3. The use of the exenatide analog as claimed in claim 1 or 2 in the preparation of drugs for the treatment of diabetes, obesity and/or complications. 4. The application according to claim 3, characterized in that the drug contains the exenatide analog as claimed in claim 1 or 2 and a pharmaceutically acceptable carrier.