CN107266556A - A kind of Africa xenopus glucagon-like peptide 1（GLP‑1）Analog and its application - Google Patents

Abstract

The present invention relates to a kind of Africa xenopus glucagon-like peptide 1（GLP‑1）Analog and application.Structural modification is carried out by the restriction enzyme site to Africa xenopus GLP 1 and C-terminal, the analogs of Africa xenopus GLP 1 with higher hypoglycemic activity and longer pharmacological action time are obtained.The solid phase synthesis process that is synthesized by of target polypeptides realizes that crude product is purified, lyophilized to obtain the analogs of Africa xenopus GLP 1.The analog synthesis yields of Africa xenopus GLP 1 of the present invention are high, cost is low, and the half-life period of analog significantly extends, and bioactivity is significantly improved.

Description

A Xenopus glucagon-like peptide-1 (GLP-1) analogue and its application

technical field

The invention relates to a Xenopus laevis glucagon-like peptide-1 (GLP-1) analog and application thereof.

Background technique

Diabetes (Diabetes Mellitus) is a chronic systemic disease related to genetic factors and various environmental factors. It is a metabolic disorder of sugar, fat and protein caused by the absolute or relative deficiency of insulin secretion in the body. It is insulin-dependent diabetes (type 1) and non-insulin-dependent diabetes (type 2), wherein type 2 patients account for more than 80% of diabetes cases. Diabetes is a global high incidence, according to the World Health Organization forecast, by 2025 the world's diabetic patients will reach 300 million. A recent epidemiological survey report in China shows that the prevalence of diabetes in China is as high as 9.7%, which is much higher than the global average prevalence of 6.4%. There are about 150 million people at potential risk of diabetes. Currently, for the treatment of type 1 diabetes, the research direction is to develop convenient and effective insulin preparations and substitutes. For the treatment of type 2 diabetes, the clinical treatment method is mainly to use oral hypoglycemic drugs. The traditional sulfonylurea and biguanide oral hypoglycemic drugs have limited efficacy. The biggest deficiency of oral diabetes drugs in the treatment of type 2 diabetes is that they cannot Reversing the etiology of diabetes, that is, "treating the symptoms without knowing the root", has no obvious effect on the growth, differentiation, and proliferation of islet β cells.

Glucagon-like peptide-1 (GLP-1) is a polypeptide incretin secreted by L cells of the small intestine. Since Mojsov discovered that GLP-1 has a strong insulin-releasing effect in 1987, relevant biological and chemical research has continued to deepen. GLP-1 can control blood sugar in patients with type 2 diabetes through a variety of mechanisms. When the blood sugar concentration rises, it can stimulate insulin secretion by highly specific binding to GLP-1 receptors, inhibit the production of glucagon, and make meals Afterwards, blood sugar decreased and remained at a constant level. Under physiological conditions, the role of GLP-1 in stimulating insulin secretion is dependent on blood glucose concentration, and hypoglycemia will not occur due to continuous secretion. In addition to regulating blood sugar, the most significant function of GLP-1 is to promote the regeneration and repair of β cells and increase the number of β cells in the pancreas. In addition, GLP-1 also has the functions of regulating nerve function, delaying gastric emptying and reducing appetite. However, endogenous or exogenous GLP-1 will be rapidly degraded by dipeptidyl peptidase IV (DPP-IV) and neutral endonuclease (NEP 24.11) in vivo and lose its biological activity, and its half-life in vivo is only two minutes So natural GLP-1 is difficult to obtain clinical use.

Although the remarkable effect of GLP-1 in the treatment of type 2 diabetes has been recognized by people, its short half-life in vivo limits the possibility of its direct drug production. DPP-IV is the main protease to inactivate natural GLP-1, and its action site is the peptide bond between Ala ⁸ -Glu ⁹ . Another main metabolic enzyme of natural GLP-1 is NEP 24.11. About 50% of GLP-1 will be degraded by NEP24.11 in the body. There are multiple enzyme cleavage sites of NEP 24.11 on the GLP-1 peptide chain, mainly Thr ¹¹ -Phe ¹² , Asp ¹⁵ -Val ¹⁶ , Ser ¹⁸ -Tyr ¹⁹ , Glu ²⁷ -Phe ²⁸ and Trp ³¹ -Leu ³² sites. Therefore, only substituting individual amino acids cannot resist the degradation of NEP24.11, while multiple amino acid substitutions may reduce the hypoglycemic activity of the compound. Most of the current research on long-acting GLP-1 receptor agonists is aimed at modifying the structure of GLP-1, which limits the development of new GLP-1 receptor agonists. At the same time, since there are many NEP 24.11 enzyme cleavage sites on natural GLP-1, it is difficult to resist the degradation of NEP24.11 by means of amino acid modification.

Therefore, it is still necessary to find GLP-1 analogs with novel structures, especially GLP-1 analogs that have certain NEP24. 1 analog. Here, we designed a new type of GLP-1 analogue based on the structure of Xenopus GLP-1, by modifying the structure of the peptide chain, so as to prolong the in vivo biological activity of the analogue and increase the hypoglycemic activity.

Contents of the invention

The present invention relates to a class of Xenopus glucagon-like peptide-1 (GLP-1) analogs, the sequence of which is:

His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Xaa2-Xaa3-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Xaa4-Xaa5-Ile-Glu-Xaa6- Xaa7-Ile-Lys-Gly-Lys-Xaa8 (SEQ. ID NO: 1)

in:

Xaa1: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa2: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa3: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa4: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa5: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa6: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa7: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser;

Xaa8: Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- _NH2 , Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys _-NH2 or _-NH2 ;

In one embodiment, the invention relates to a Xenopus GLP-1 analogue having the following sequence:

His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys- _NH2 (SEQ. ID NO: 2)

In one embodiment, the invention relates to a Xenopus GLP-1 analogue having the following sequence:

His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys- _NH2 (SEQ. ID NO: 3)

In one embodiment, the invention relates to a Xenopus GLP-1 analogue having the following sequence:

His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser- _NH2 (SEQ. ID NO: 4)

In one embodiment, the invention relates to a Xenopus GLP-1 analogue having the following sequence:

His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser- _NH2 (SEQ. ID NO: 5)

In one embodiment, the invention relates to a Xenopus GLP-1 analogue having the following sequence:

His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- _NH2 (SEQ.ID NO: 6)

In one embodiment, the invention relates to a Xenopus GLP-1 analogue having the following sequence:

His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- _NH2 (SEQ. ID NO: 7)

The present invention also provides a pharmaceutical composition, comprising a therapeutically effective amount of at least one compound and its pharmaceutically acceptable salt, or a pharmaceutically acceptable carrier or diluent.

The present invention further provides the use of the above compound and its pharmaceutically acceptable salt, or a pharmaceutically acceptable carrier or diluent in the preparation of drugs for diabetes or weight loss.

The chemical properties of the above-mentioned compounds provided by the present invention are stable and difficult to be degraded by dipeptidyl peptidase IV (DPP-IV) and neutral endonuclease in the body. 1 Must be continuous intravenous infusion or continuous subcutaneous injection to produce the defect of curative effect. In addition, when the above-mentioned compound provided by the present invention or the pharmaceutical composition prepared from the compound as an active ingredient is used for blood sugar lowering and weight loss treatment, it not only has a significant long-term hypoglycemic effect, but also has an obvious weight loss effect.

The present invention also provides a preparation method for the above-mentioned compound. The present invention adopts the Fmoc/tBu orthogonal protection solid-phase synthesis strategy to obtain the above-mentioned target compound through high-efficiency and high-yield synthesis.

The advantages of the present invention are:

1. A Xenopus laevis glucagon-like peptide-1 (GLP-1) analog is proposed, which is a new type of GLP-1 analog with a new structure, which helps to promote the development of new GLP-1 drugs research and development.

2. The peptide chain of the Xenopus GLP-1 analog itself has good NEP 24.11 resistance, and it does not need to be chemically modified like natural GLP-1 to achieve resistance to NEP 24.11 enzymatic degradation, thereby avoiding chemical Problems such as hypoglycemic activity and water solubility reduction brought about by modification.

3. The biological half-life of the Xenopus laevis GLP-1 analogue involved in the present invention is significantly longer than that of natural GLP-1, and its hypoglycemic activity is also significantly better than that of natural GLP-1. In addition, it also has a good weight loss effect , can be used in the treatment of diabetes and obesity.

4. The synthetic process of the Xenopus laevis GLP-1 analog involved in the present invention is simple, the cost is low, and the purity of the crude product is greater than 80%, which is greatly improved compared with the conventional solid-phase synthesis method, and it is easy to realize automation and large-scale production, which makes it more Suitable for industrial production.

Therefore, the Xenopus laevis GLP-1 analogue provided by the present invention has short synthesis period, high yield, easy crude product purification, low production cost and easy industrial automation production. The prepared Xenopus laevis GLP-1 analogue is more stable than natural GLP-1, its hypoglycemic action time and hypoglycemic activity are better than natural GLP-1, and it also has a good weight loss effect, and is suitable as a treatment for diabetes and Active ingredient in weight loss drugs.

Description of drawings

The invention has been generally described above, and the following drawings are used to illustrate specific embodiments of the invention. in:

Figure 1 shows the temperature degradation diagram of GLP-1 and Xenopus GLP-1 analogs in NEP 24.11 enzyme;

Figure 2 shows the warm compress degradation diagram of GLP-1 and Xenopus GLP-1 analog in rat plasma;

Figure 3 shows the blood glucose chart of the abdominal cavity glucose tolerance test of GLP-1 and Xenopus GLP-1 analogs in db/db model mice;

Figure 4 shows the blood glucose chart of GLP-1 and Xenopus GLP-1 analogs in db/db model mice administered in advance in the intraperitoneal glucose tolerance test;

Figure 5 shows the weight gain of mice in long-term treatment experiments with Exenatide and Xenopus GLP-1 analogs;

detailed description

The following abbreviations are used throughout this specification:

DMF: dimethylformamide; DCM: dichloromethane; Fmoc: N-9-fluorenylmethoxycarbonyl; DIC: N,N'-diisopropylcarbodiimide; HOBT: 1-hydroxy-benzotri Azole; TFA: trifluoroacetic acid; EDT: ethanedimercapto; HPLC: high performance liquid chromatography; ESI-MS: electrospray mass spectrometry; LC-MS: liquid chromatography-mass spectrometry; Gly: glycine; Ser: serine; Ala : alanine; Thr: threonine; Val: valine; Ile: isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: histidine; Pro : Proline; Asp: Aspartic Acid; Met: Methionine; Glu: Glutamic Acid; Trp: Tryptophan; Lys: Lysine; Arg: Arginine; Asn: Asparagine; Gln: Glutamine Amide; Cys: cysteine.

The present invention is illustrated by the following examples, but these examples are not to be construed as limiting the invention in any way.

Example 1

His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys- _NH2 (SEQ. ID NO: 2)

Solid-phase synthesis of (0.1mmol)

(1) Swelling of the resin

Weigh 0.262g of Fmoc-Rink amide-MBHA Resin (substitution amount 0.382mmol/g), swell with 7mL DCM for 2min, remove DCM by suction filtration, then swell with 10mL DCM for 30min, and finally rinse with DCM and DMF 7mL respectively.

(3) Removal of Fmoc protecting group

Put the swollen resin into the reactor, add 7mL of 20% piperidine/DMF (V/V) solution, shake back and forth at 180°C for 5min in the reactor, the reaction temperature is 25°C, and filter the solution after the reaction; Add 7 mL of 20% piperidine/DMF (V/V) solution and shake back and forth at 180°C for 15 min in the reactor at a reaction temperature of 25°C. After the reaction, the solution was filtered off and washed with DMF. A resin free of the initially attached Fmoc protecting group is obtained.

(4) Synthesis of Lys-Rink amide-MBHA Resin

Dissolve Lys (0.4mmol), DIC (0.4mmol), HOBT (0.4mmol) in 5mL DMF, then add this solution to the above resin, shake back and forth at 180 degrees in the reactor for 90min, and the reaction temperature is 25°C . After the reaction was completed, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL of DMF.

(5) Detection of coupling efficiency

Use the ninhydrin method to detect the coupling efficiency of the resin, and if the color reaction is negative, you can enter the next coupling cycle.

Ninhydrin method: Take a small amount of resin particles and wash them with ethanol, put them into a transparent vial, add 5% ninhydrin ethanol, KCN pyridine solution (2ml 0.001M KCN diluted in 98ml pyridine), 2 drops each of 80% phenol ethanol solution, Heat at 100°C for 5 minutes, if the resin turns blue, it is positive.

(6) Extension of the peptide chain

According to the sequence of the peptide chain, the above steps of deprotection and coupling are repeated to connect the corresponding amino acids in sequence until the synthesis of the peptide chain is completed, and the resin with the compound connected is obtained.

(7) Cleavage of polypeptides on the resin

Put the compound-linked resin obtained above into a reaction flask, and add 5 mL of cracking agent Reagent K (TFA/sulfide anisole/water/phenol/EDT, 82.5:5:5:5:2.5, V/V) , and then stirred at room temperature for 2 h. After the reaction was completed, the mixture was filtered with suction, washed three times with a small amount of TFA, the filtrates were combined, and the filtrates were concentrated under reduced pressure. The concentrated filtrate was added to a large amount of glacial ether to precipitate a white flocculent precipitate, which was refrigerated and centrifuged to obtain the crude product of the target polypeptide with a final yield of 91.9%.

(8) Purification of polypeptides

The crude polypeptide was dissolved in 50% methanol/water, and purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse-phase column (320mm×28mm, 5 μm); mobile phase A: 0.1% TFA/water (V/V), Mobile phase B: methanol (V/V); mobile phase gradient: mobile phase B 30% to 90%, 20min; flow rate is 10mL/min, detection wavelength is 214nm, the collected solution is decompressed to remove methanol, freeze-dried to obtain pure product peptide. The theoretical relative molecular mass is 3513.9. ESI-MS m/z: calu [M+3H] ³⁺ 1172.3, [M+4H] ⁴⁺ 879.5; found [M+3H] ³⁺ 1172.5, [M+4H] ⁴⁺ 879.6.

Embodiment 2～6

According to the method described in Example 1, the Xenopus laevis glucagon-like peptide-1 (GLP-1) analogues of Examples 2-6 were synthesized according to the corresponding sequences, and their molecular weights were confirmed by ESI-MS.

Example 2

His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys- _NH2 (SEQ. ID NO: 3)

The theoretical relative molecular mass is 3499.9. ESI-MS m/z: calu [M+3H] ³⁺ 1167.6, [M+4H] ⁴⁺ 875.9; found [M+3H] ³⁺ 1167.7, [M+4H] ⁴⁺ 876.2;

Example 3

His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser- _NH2 (SEQ. ID NO: 4)

The theoretical relative molecular mass is 4291.7. ESI-MS m/z: calu [M+3H] ³⁺ 1431.6, [M+4H] ⁴⁺ 1073.9; found [M+3H] ³⁺ 1431.2, [M+4H] ⁴⁺ 1073.5.

Example 4

His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser- _NH2 (SEQ. ID NO: 5)

The theoretical relative molecular mass is 4277.7. ESI-MS m/z: calu [M+3H] ³⁺ 1426.9, [M+4H] ⁴⁺ 1070.4; found [M+3H] ³⁺ 1427.4, [M+4H] ⁴⁺ 1070.8.

Example 5

His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- _NH2 (SEQ.ID NO: 6)

The theoretical relative molecular mass is 4963.7. ESI-MS m/z: calu [M+4H] ⁴⁺ 1241.9, [M+5H] ⁵⁺ 993.7; found [M+4H] ⁴⁺ 1241.8, [M+5H] ⁵⁺ 993.4.

Example 6

His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- _NH2 (SEQ. ID NO: 7)

The theoretical relative molecular mass is 4949.7. ESI-MS m/z: calu [M+4H] ⁴⁺ 1238.4, [M+5H] ⁵⁺ 990.9; found [M+4H] ⁴⁺ 1238.7, [M+5H] ⁵⁺ 991.4.

Example 7

Stability Experiment of Xenopus GLP-1 Analogs to NEP 24.11

Purified Xenopus GLP-1 analogs and natural GLP-1 (control) 5nmol and 5mU NEP 24.11 were incubated in 200 μL of 50mM Tris-HCL buffer solution at 37°C for 12h, pH 7.4. Finally, 10 μL of 20% acetonitrile/water solution was added to stop the reaction. Take the incubation solution at 0, 1, 2, 4, 6, and 12 hours respectively, centrifuge, take the supernatant, and analyze it by LC-MS, calculate the peak area at each time point, and draw the decay curve. The analysis adopts C18 reverse phase column (150mm×4.6mm, 5μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); mobile phase gradient: mobile Phase B 10%-45%, 22min; flow rate 0.3mL/min; column temperature 30°C; detection wavelength 214nm.

As shown in Figure 1, after incubation with NEP 24.11 for 4 hours, the unmodified natural GLP-1 was basically hydrolyzed, and the complete peptide chain was less than 10%. However, the intact peptide chain of Xenopus laevis GLP-1 analog was more than 80% after incubation with DPP-IV for 12 hours. The results showed that the Xenopus GLP-1 analogs we designed could resist the enzymatic hydrolysis of NEP 24.11.

Example 8

Stability Experiment of Xenopus GLP-1 Analogues in Rat Plasma

The blood was collected from the eyeball of the rat, and the blood was put into a centrifuge tube containing heparin, centrifuged at 3000rpm for 10 minutes, and the supernatant plasma was taken as the incubation plasma, and the response signal of the compound was detected by LC-MS. 100ul of GLP-1 (control) and Xenopus GLP-1 analogs and 100ul of plasma, vortexed and mixed, placed in a 37°C water bath, incubated for 24 hours, at 0, 1, 2, 4, 8, 12 , Take 10ul at 24h time point, add 20ul acetonitrile to precipitate, centrifuge at 14000rpm, take the supernatant into LC-MS, calculate the peak area at each time point, and draw the decay curve.

As shown in Figure 2, the plasma half-life of the unmodified prototype GLP-1 is only about 15 minutes, while the plasma half-life of Xenopus GLP-1 analogs is more than 10 hours.

Example 9

Intraperitoneal glucose tolerance test of Xenopus laevis GLP-1 analog

Six-week-old db/db diabetic model mice were adaptively fed for one week and randomly divided into six groups. After fasting for 18 hours, the mice were given positive control GLP-1 (25nmol/kg) intraperitoneally 5 minutes in advance, negative control saline and Xenopus GLP-1 analog (25nmol/L), and glucose (1g/kg) was given intraperitoneally for 0 minutes , Monitor blood sugar levels with a blood glucose meter at 0, 15, 30, 60, and 120 minutes.

As shown in Figure 3, the hypoglycemic activity of the Xenopus GLP-1 analog is better than that of natural GLP-1, indicating that the Xenopus GLP-1 analog has a strong ability to control blood sugar.

Example 10

Intraperitoneal glucose tolerance test of Xenopus laevis GLP-1 analogue administered in advance

Six-week-old db/db diabetic model mice were adaptively fed for one week and randomly divided into six groups. After fasting for 18 hours, the mice were given positive control GLP-1 (25nmol/kg) intraperitoneally 120 minutes in advance, negative control saline and Xenopus GLP-1 analogue (25nmol/L), and glucose (1g/kg) was given intraperitoneally for 0 minutes , Monitor blood sugar levels with a blood glucose meter at 0, 15, 30, 60, and 120 minutes. After the end of the first abdominal glucose tolerance cycle, intraperitoneal injection of glucose was repeated twice at 6 and 12 hours, and the detection interval of blood glucose in each glucose tolerance cycle was the same as the first time.

As shown in Figure 4, after administration 120 minutes in advance, natural GLP-1 did not show hypoglycemic activity throughout the glucose tolerance test, indicating that GLP-1 has been completely metabolized. However, the hypoglycemic activity of the Xenopus GLP-1 analog has not changed, indicating that the analog has high stability in vivo and has long-term hypoglycemic activity.

Example 11

Long-term therapeutic experiments with Xenopus GLP-1 analogues

6-week-old db/db diabetic model mice were adaptively fed for one week and randomly divided into 6 mice in each group. The mice were given the positive control Exenatide (25nmol/kg) and the negative control saline twice a day, and the treatment group was given the Xenopus laevis GLP-1 analog (25nmol/L) once a day, and the treatment cycle was 20 days, every two days Check the body weight of the mice once.

As shown in Figure 5, the Xenopus GLP-1 analogue can significantly inhibit the weight gain of mice, and its body weight loss effect is better than that of the positive control Exenatide, indicating that the Xenopus GLP-1 analogue has a good therapeutic effect on diabetes and Medicinal prospects for weight loss.

<110> Jiangsu Normal University

<120> A Xenopus glucagon-like peptide-1 (GLP-1) analogue and its application

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<210> 1

<211> 31

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<213> Artificial sequence

<220>

<221> Synthetic constructs

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<221> Synthetic constructs

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<221> Synthetic constructs

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<221> Synthetic constructs

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<220>

<221> Synthetic constructs

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<220>

<221> Synthetic constructs

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His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu Lys Ala Ala

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His Gly Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu Lys Ala Ala

1 5 10 15

Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Pro Ser Ser Gly Ala Pro Pro Pro Ser

20 25 30 35

<210> 6

<211> 44

<212> PRT

<213> Artificial sequence

<220>

<221> Synthetic constructs

His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu Lys Ala Ala

1 5 10 15

Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Pro Ser Ser Gly Ala Pro Ser Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30 35 40

<210> 7

<211> 44

<212> PRT

<213> Artificial sequence

<220>

<221> Synthetic constructs

His Gly Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu Lys Ala Ala

1 5 10 15

Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Pro Ser Ser Gly Ala Pro Ser Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30 35 40

Claims (7)

1. A Xenopus laevis glucagon-like peptide-1 (GLP-1) analog containing the structure of formula I (SEQ.ID NO: 1), whose sequence is: His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Xaa2-Xaa3-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Xaa4-Xaa5-Ile-Glu-Xaa6- Xaa7-Ile-Lys-Gly-Lys-Xaa8 (SEQ. ID NO: 1) in: Xaa1: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa2: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa3: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa4: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa5: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa6: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa7: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser; Xaa8: Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2, Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- NH2 or -NH2. 2. Xenopus laevis glucagon-like peptide-1 (GLP-1) analogue according to claim 1, preferred sequence is: His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys- _NH2 (SEQ. ID NO: 2) His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys- _NH2 (SEQ. ID NO: 3) His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser- _NH2 (SEQ. ID NO: 4) His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser- _NH2 (SEQ. ID NO: 5) His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- _NH2 (SEQ.ID NO: 6) His-Gly-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp- Leu-Ile-Lys-Gly-Lys-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys- _NH2 (SEQ. ID NO: 7). 3. A pharmaceutical composition comprising a therapeutically effective amount of at least one Xenopus glucagon-like peptide-1 (GLP-1 ) analogue as claimed in claim 1 and pharmaceutically acceptable salts thereof. 4. A pharmaceutical composition comprising a therapeutically effective amount of at least one Xenopus glucagon-like peptide-1 (GLP-1) analogue described in claim 1 and a pharmaceutically acceptable carrier or diluent agent. 5. A Xenopus laevis glucagon-like peptide-1 (GLP-1) analogue as claimed in claim 3 and its pharmaceutically acceptable salt are used in the preparation of medicines for diabetes or weight loss . 6. A Xenopus laevis glucagon-like peptide-1 (GLP-1) analog as claimed in claim 4 and a pharmaceutically acceptable carrier or diluent are used in the preparation of medicines for diabetes or weight loss use. 7. A method for preparing the Xenopus laevis glucagon-like peptide-1 (GLP-1) analog as claimed in claim 1, comprising biological expression, liquid phase synthesis and solid phase synthesis preparation methods.