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

A kind of Africa xenopus glucagon-like peptide 1（GLP‑1）Analog and its application Download PDF

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CN107266556A
CN107266556A CN201611156564.XA CN201611156564A CN107266556A CN 107266556 A CN107266556 A CN 107266556A CN 201611156564 A CN201611156564 A CN 201611156564A CN 107266556 A CN107266556 A CN 107266556A
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glu
ala
gly
thr
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韩京
傅俊杰
崔旭
周凤
费颖颖
张莹
司鹏斌
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Jiangsu Normal University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides

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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 kind of Africa xenopus glucagon-like-peptide-1 (GLP-1) analog and its application
Technical field
The present invention relates to a kind of Africa xenopus glucagon-like-peptide-1 (GLP-1) analog and its application.
Background technology
Diabetes (Diabetes Mellitus) are a kind of relevant with inherent cause and related to a variety of environmental factors Chronic systemic disease, is due to sugar caused by the absolute or relative deficiency of internal insulin secretion, fat, the generation of protein Thank to disorder, insulin-dependent diabetes mellitus (1 type) and Non-Insulin Dependent Diabetes Mellitus (2 type) can be divided into, wherein 2 type patients account for More than the 80% of diabetes cases.Diabetes are a kind of global high morbidities, are predicted according to the World Health Organization, complete by 2025 World diabetic is up to 300,000,000.The recent report on Epidemiological of China shows that diabetes mellitus in China illness rate is up to 9.7%, far above the average illness rate 6.4% in the whole world, estimate that China's diabetes number of patients is more than 9000 by this illness rate Ten thousand, and there is also about 1.5 hundred million potential risk of diabetes crowds.At present for the treatment of type 1 diabetes, research direction is exploitation Convenient drug administration, effective insulin preparation and substitute.And for the treatment of diabetes B, clinical treatment means, which are mainly, to be made With oral hypoglycemic drug, traditional sulfonylurea and biguanides OHA offer limited effectiveness, oral diabetes drug is in treatment 2 In patients with type Ⅰ DM disease maximum deficiency be can not reverting diabetes the cause of disease, i.e., " take stopgap measures do not know this ", for the life of beta Cell of islet Long, differentiation, propagation are without obvious effect.
Glucagon-like-peptide-1 (glucagon like peptide-1, GLP-1) is one kind of Intestinal L cells secretion Polypeptide incretin.Since finding that GLP-1 has strong pancreotropic hormone release action from Mojsov in 1987, relevant life Thing and chemical research deepen continuously.GLP-1 can control the blood glucose of diabetes B patient by number of mechanisms, work as blood sugar concentration During rise, by specifically being combined with GLP-1 high receptors, stimulate insulin secretion, the generation of glucagon suppression makes Postprandial blood sugar reduces and maintains constant level.In physiological conditions, the effect that GLP-1 stimulates insulin secretion depends on blood glucose Concentration, will not occur hypoglycemia because of continuous release.Except regulation blood glucose, the most significant functions of GLP-1 are to promote β cells again Raw and reparation, increases beta Cell of islet quantity.In addition GLP-1 also has regulation nervous function, delay gastric emptying and reduction appetite etc. Effect.But endogenous or exogenous GLP-1 in vivo can be rapidly by DPP IVs (DPP-IV) and neutral restriction endonuclease (NEP 24.11) degrades and loses bioactivity, and its Half-life in vivo only has two minutes or so, therefore natural GLP-1 is difficult to obtain Clinical practice.
Although remarkable results of the GLP-1 in diabetes B treatment is recognized by people, it in vivo too short half The phase of declining limits the possibility of its direct patent medicine.DPP-IV is to make the major protein enzyme of natural GLP-1 inactivations, and its action site is Ala8-Glu9Between peptide bond.Natural GLP-1 another main metabolic enzyme is NEP 24.11, and about 50% GLP-1 is in vivo It can be degraded by NEP24.11, there is multiple NEP 24.11 restriction enzyme site, mainly Thr on GLP-1 peptide chains11-Phe12、Asp15- Val16、Ser18-Tyr19、Glu27-Phe28And Trp31-Leu32Site.Therefore, only displacement Individual amino acids can not resist NEP 24.11 degradeds, and multiple amino acid replacements may decline the hypoglycemic activity of compound.At present major part long-acting GLP-1 by The research of body activator is modified both for GLP-1 structure, and which has limited the research and development of the receptor stimulating agent of novel glp-1-1. Simultaneously as there is the more restriction enzyme sites of NEP 24.11 on natural GLP-1, it is difficult to resist NEP by amino acid modified means 24.11 degraded.
Therefore, it is still necessary to find the GLP-1 analogs with new structure, particularly inherently there is certain NEP The GLP-1 analogs of 24.11 resistances and hypoglycemic activity, so as to find the analog of novel glp-1-1 of efficient, the long-acting hypoglycemic of energy.This In, we devise a kind of analog of novel glp-1-1 based on Africa xenopus GLP-1 structures, are repaiied by carrying out structure to peptide chain Decorations, so as to extend in vivo bioactivity action time and the increase hypoglycemic activity of analog.
The content of the invention
The present invention relates to class Africa xenopus glucagon-like-peptide-1 (GLP-1) analog, its 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)
Wherein：
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-NH2Or-NH2；
In one embodiment, the present invention relates to the Africa xenopus GLP-1 analogs with 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 present invention relates to the Africa xenopus GLP-1 analogs with 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 present invention relates to the Africa xenopus GLP-1 analogs with 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-Ser-NH2(SEQ.ID NO：4)
In one embodiment, the present invention relates to the Africa xenopus GLP-1 analogs with 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-Ser-NH2(SEQ.ID NO：5)
In one embodiment, the present invention relates to the Africa xenopus GLP-1 analogs with 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 present invention relates to the Africa xenopus GLP-1 analogs with 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)
Present invention also offers at least one above-claimed cpd and its medicine of a kind of pharmaceutical composition, including therapeutically effective amount Acceptable salt on, or pharmaceutically acceptable carrier or diluent.
Invention further provides above-claimed cpd and its pharmaceutically acceptable salt, or pharmaceutically acceptable carrier Or diluent is being prepared for the utilization in diabetes or slimming medicine.
Above-claimed cpd chemical property that the present invention is provided is stable, be difficult by internal DPP IV (DPP-IV) and Neutral endonucleases, the internal blood sugar reducing function time of all compounds significantly extends, and overcomes natural GLP-1 and has to last for Drip-feed or continuous subcutaneous infusion could produce the defect of curative effect.In addition, above-claimed cpd or compound that the present invention is provided When the pharmaceutical composition prepared as active ingredient is used to reduce blood glucose and weight reducing treatments, existing significant long-acting hypoglycemic effect, Also obvious antiobesity action.
Present invention also offers the preparation method of above-claimed cpd, the present invention uses the orthogonal protection synthesis in solid state of Fmoc/tBu Tactful efficient, synthesis in high yield obtains above-mentioned target compound.
The advantage of the invention is that：
1st, a kind of Africa xenopus glucagon-like-peptide-1 (GLP-1) analog proposed, is that a class has brand new The analog of novel glp-1-1, help lend some impetus to the research and development of the class medicine of novel glp-1-1.
2nd, the peptide chain of Africa xenopus GLP-1 analogs inherently has the good resistances of NEP 24.11, it is not necessary to as natural GLP-1 must be equally chemically modified to it could realize the resistance to enzymes of NEP 24.11 degraded, so as to avoid chemical modification institute band The problems such as hypoglycemic activity come and water-soluble decline.
3rd, Africa xenopus GLP-1 analogs involved in the present invention, its biological half-life, more natural GLP-1 significantly extended, Its hypoglycemic activity is also significantly better than natural GLP-1, in addition, it also has good fat-reducing effect, available for diabetes and obesity Treatment.
4th, Africa xenopus GLP-1 analogs building-up process involved in the present invention is simple, and cost is low, and crude product purity is more than 80%, more conventional solid phase synthesis process is greatly improved, and easy to automate, large-scale, and this makes it be more suitable for industry Metaplasia is produced.
Therefore the Africa xenopus GLP-1 analogs that the present invention is provided, synthesis cycle is short, high income, purifying crude easy, raw Production cost is low, be easy to industrial automation production.The Africa xenopus GLP-1 analogs prepared are more steady than natural GLP-1 Fixed, hypoglycemic effect time and hypoglycemic activity are better than natural GLP-1, and also have good fat-reducing effect, are suitable as Treat the active component of diabetes and slimming medicine.
Brief description of the drawings
General description is done to the present invention above, accompanying drawings below is used to illustrate specific embodiments of the present invention.Wherein：
Fig. 1 is shown GLP-1 and applies degraded figure with temperature of the Africa xenopus GLP-1 analogs in the enzymes of NEP 24.11；
Fig. 2 is shown GLP-1 and applies degraded figure with temperature of the Africa xenopus GLP-1 analogs in rat plasma；
GLP-1 and abdominal cavity sugar tolerance of the Africa xenopus GLP-1 analogs in db/db model mice bodies is shown in Fig. 3 Test blood glucose figure；
GLP-1 and in advance administration abdomen of the Africa xenopus GLP-1 analogs in db/db model mice bodies is shown in Fig. 4 Chamber sugar tolerance tests blood glucose figure；
Exenatide is shown in Fig. 5, and the body weight of mouse in long-term treatment experiment increases with Africa xenopus GLP-1 analogs Dosage；
Embodiment
Following abbreviation is used in this specification：
DMF：Dimethylformamide；DCM：Dichloromethane；Fmoc：N-9- fluorenylmethyloxycarbonyls；DIC：N, N '-diisopropyl carbon Diimine；HOBT：1- hydroxyls-BTA；TFA：Trifluoroacetic acid；EDT：Dimercaptoethane；HPLC：High performance liquid chromatography； ESI-MS：Electrospray ionization mass spectrum；LC-MS：LC-MS mass spectrum；Gly：Glycine；Ser：Serine；Ala：Alanine；Thr：Soviet Union Propylhomoserin；Val：Valine；Ile：Isoleucine；Leu：Leucine；Tyr：Tyrosine；Phe：Phenylalanine；His：Histidine； Pro：Proline；Asp：Asparatate；Met：Methionine；Glu：Glutamic acid；Trp：Tryptophan；Lys：Lysine；Arg：Essence Propylhomoserin；Asn：Asparagine；Gln：Glutamine；Cys：Cysteine.
The present invention is illustrated by the following example, but these embodiments do not do the solution of any limitation present invention Release.
Embodiment 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)
Synthesis in solid state (0.1mmol)
(1) resin is swelled
Fmoc-Rink amide-MBHA Resin 0.262g (substitution amount 0.382mmol/g) are weighed, it is molten through 7mL DCM Swollen 2min, suction filtration removes DCM, then is swelled 30min with 10mL DCM, finally uses DCM, DMF 7mL to rinse well respectively.
(3) removing of Fmoc protection groups
The resin being swelled is put into reactor, 20% piperidines of 7mL/DMF (V/V) solution is added, in the reactor 180 degree comes and goes shaking reaction 5min, and 25 DEG C of reaction temperature, reaction filters off solution after terminating；Add 20% piperidines of 7mL/DMF (V/V) 180 degree comes and goes shaking reaction 15min, 25 DEG C of reaction temperature to solution in the reactor.Reaction filters off solution after terminating, and uses DMF washes cleans.Obtain sloughing the resin of the Fmoc protection groups initially connected.
(4) Lys-Rink amide-MBHA Resin synthesis
By Lys (0.4mmol), DIC (0.4mmol), HOBT (0.4mmol) is dissolved in 5mL DMF, then this solution is added In resin above, 180 degree comes and goes shaking reaction 90min, 25 DEG C of reaction temperature in the reactor.Reaction filters out reaction after terminating Liquid, resin is washed with 7mL DMF 3 times.
(5) detection of coupling efficiency
The coupling efficiency of resin is detected with ninhydrin method, chromogenic reaction is that feminine gender can enter next coupling cycles.
Ninhydrin method：Take a small amount of resin particle to be washed with ethanol, be put into transparent vials and add 5% ninhydrin ethanol, KCN Each 2 drop of pyridine solution (2ml 0.001M KCN are diluted in 98ml pyridines), 80% phenol ethanol solution, 5 points are heated in 100 DEG C Clock, if the aobvious blueness of resin is the positive.
(6) extension of peptide chain
According to the sequence of peptide chain, repeat above-mentioned deprotection and be sequentially connected corresponding amino acid the step of coupling until peptide Chain synthesis is finished, and obtains being connected with the resin of compound.
(7) on resin polypeptide cracking
The resin obtained above for being connected with compound is put into reaction bulb, it is each to add decomposition agent Reagent K (TFA/ benzene Methyl sulfide/water/phenol/EDT, 82.5: 5: 5: 5: 2.5, V/V) 5mL, then stirring reaction 2h at normal temperatures.Reaction is taken out after terminating Filter, plus a small amount of TFA are washed three times, merging filtrate, filtrate decompression concentration.It will be separated out in vain in the substantial amounts of ice ether of concentration filtrate addition Color flocculent deposit, refrigerated centrifuge obtains the crude product of target polypeptides, and ultimate yield is 91.9%.
(8) purifying of polypeptide
Crude product polypeptide is dissolved in 50% methanol/water, purified using preparative liquid chromatography, chromatographic condition is：C18 is anti-phase Post (320mm × 28mm, 5 μm)；Mobile phase A：0.1%TFA/ water (V/V), Mobile phase B：Methanol (V/V)；Eluent gradient：Stream Dynamic phase B 30%~90%, 20min；Flow velocity is 10mL/min, and Detection wavelength is 214nm, and the solution decompression of collection removes methanol, Freeze-drying obtains sterling polypeptide.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
Method according to embodiment 1 is high according to the Africa xenopus pancreas that corresponding sequent synthesis obtains embodiment 2~6 (GLP-1) analog of blood glucose element sample peptide -1, respective molecular weight is confirmed by ESI-MS.
Embodiment 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)
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；.
Embodiment 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-Ser-NH2(SEQ.ID NO：4)
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。
Embodiment 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-Ser-NH2(SEQ.ID NO：5)
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。
Embodiment 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)
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。
Embodiment 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)
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。
Embodiment 7
Stability experiment of the Africa xenopus GLP-1 analogs to NEP 24.11
By Africa xenopus GLP-1 analogs after purification and natural GLP-1 (control) 5nmol and 5mU NEP 24.11 In 200 μ L concentration in 50mM Tris-HCL cushioning liquid, 37 DEG C of temperature incubate 12h, pH 7.4.It is eventually adding 10 μ L's 20% Acetonitrile/water solution terminating reaction.Take the temperature of 0,1,2,4,6,12h point to incubate solution respectively, centrifuge, take supernatant, enter LC-MS points Analysis, calculates the peak area of Each point in time, makes attenuation curve.Analysis is using C18 reversed-phase columns (150mm × 4.6mm, 5 μm)；Stream Dynamic phase A：0.1%TFA/ water (V/V), Mobile phase B：0.1%TFA/ acetonitriles (V/V)；Eluent gradient：Mobile phase B 10%~ 45%, 22min；Flow velocity is 0.3mL/min；Column temperature is 30 DEG C；Detection wavelength is 214nm.
As shown in figure 1, not engineered natural GLP-1 is substantially all hydrolyzed after 4h is incubated with the temperature of NEP 24.11, it is complete Whole peptide chain is less than 10%.And Africa xenopus GLP-1 analogs incubate peptide chain complete after 12h more than 80% with DPP-IV temperature.Knot Fruit shows that the Africa xenopus GLP-1 analogs that we design have resistance NEP 24.11 enzymolysis.
Embodiment 8
Stability experiment of the Africa xenopus GLP-1 analogs to rat plasma
Rat eye takes blood, and blood is fitted into the centrifuge tube containing heparin, and 3000rpm is centrifuged 10 minutes, takes supernatant blood plasma Blood plasma is incubated as temperature, using LC-MS come the response signal of detection compound.100ul GLP-1 (control) and Africa xenopus GLP- Inserted after the blood plasma of 1 analog and 100ul, vortex mixed in 37 DEG C of water-baths, temperature is incubated 24 hours, at 0,1,2,4,8,12,24h Between point take 10ul, add 20ul acetonitrile precipitations, 14000rpm centrifugations take supernatant to enter LC-MS, calculate the peak face of Each point in time Product, makes attenuation curve.
As shown in Fig. 2 not the plasma half-life of the prototype GLP-1 by transformation there was only 15min or so, and Africa xenopus The plasma half-life of GLP-1 analogs is all more than 10 hours.
Embodiment 9
The abdominal cavity sugar tolerance experiment of Africa xenopus GLP-1 analogs
6 week old db/db diabetic mices, adaptability is raised one week, random packet, every group six.Mouse is in fasting 18h, in advance 5min abdominal cavities give positive control GLP-1 (25nmol/kg), negative control physiological saline and Africa xenopus GLP-1 Analog (25nmol/L), 0min gives in abdominal cavity glucose (1g/kg), and blood is monitored with blood glucose meter 0,15,30,60,120min Sugar level.
As shown in figure 3, the hypoglycemic activity of Africa xenopus GLP-1 analogs is better than natural GLP-1, illustrate Africa xenopus GLP- 1 analog has very strong glycemic control.
Embodiment 10
The abdominal cavity of the administration in advance sugar tolerance experiment of Africa xenopus GLP-1 analogs
6 week old db/db diabetic mices, adaptability is raised one week, random packet, every group six.Mouse is in fasting 18h, in advance 120min abdominal cavities give positive control GLP-1 (25nmol/kg), negative control physiological saline and Africa xenopus GLP- 1 analog (25nmol/L), 0min gives in abdominal cavity glucose (1g/kg), and blood is monitored with blood glucose meter 0,15,30,60,120min Sugar level.After first time abdominal cavity sugar tolerance end cycle, intraperitoneal injection glucose, each sugar tolerance week are repeated twice in 6 and 12h The assay intervals of phase blood glucose are identical with first time.
As shown in figure 4, after 120min administrations in advance, natural GLP-1 does not show hypoglycemic in the experiment of whole sugar tolerance Activity, illustrates that GLP-1 is metabolized completely.And the hypoglycemic activity of Africa xenopus GLP-1 analogs does not change, illustrate class Internal stability like thing is very high, with long-acting hypoglycemic activity.
Embodiment 11
The long-term treatment experiment of Africa xenopus GLP-1 analogs
6 week old db/db diabetic mices, adaptability is raised one week, random packet, every group 6.Mouse is 2 times a day Give positive control Exenatide (25nmol/kg), negative control physiological saline, administration group 1 day gives Africa xenopus 1 time GLP-1 analogs (25nmol/L), treatment cycle 20 days, the body weight of the mouse of detection in every two days.
As shown in figure 5, Africa xenopus GLP-1 analogs can significantly inhibit the body weight increase of mouse, and its body weight subtracts Catheresis is better than positive control Exenatide, and indicating Africa xenopus GLP-1 analogs has good treatment diabetes and subtract The prospect in medicine of fertilizer.
<110>Jiangsu Normal University
<120>A kind of Africa xenopus glucagon-like-peptide-1（GLP-1）Analog and its application
<160> 6
<210> 1
<211> 31
<212> PRT
<213>Artificial sequence
<220>
<221>Synthesize construct
<222> (2)..(2)
<223>The Xaa of the 2nd is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (9)..(9)
<223>The Xaa of the 9th is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (10)..(10)
<223>The Xaa of the 10th is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (21)..(21)
<223>The Xaa of the 21st is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (22)..(22)
<223>The Xaa of the 22nd is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (25)..(25)
<223>The Xaa of the 25th is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (26)..(26)
<223>The Xaa of the 26th is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Val, Tyr, Thr, Trp or Ser
<220>
<221>Synthesize construct
<222> (31)..(31)
<223>The Xaa of the 31st is 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
<400> 1
His Xaa Glu Gly Thr Tyr Thr Asn Xaa Xaa Thr Glu Tyr Leu Glu Glu Lys Ala Ala
1 5 10 15
Lys Xaa Xaa Ile Glu Xaa Xaa Ile Lys Gly Lys Xaa
20 25 30
<210> 2
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<221>Synthesize construct
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
20 25 30
<210> 3
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<221>Synthesize construct
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
20 25 30
<210> 4
<211> 39
<212> PRT
<213>Artificial sequence
<220>
<221>Synthesize construct
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 Pro Pro Ser
20 25 30 35
<210> 5
<211> 39
<212> PRT
<213>Artificial sequence
<220>
<221>Synthesize construct
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>Synthesize construct
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 Pro Ser Lys Lys Lys Lys Lys Lys
20 25 30 35 40
<210> 7
<211> 44
<212> PRT
<213>Artificial sequence
<220>
<221>Synthesize construct
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 Ser Lys Lys Lys Lys Lys Lys
20 25 30 35 40

Claims (7)

1. one kind contains Formulas I (SEQ.ID NO：1) Africa xenopus glucagon-like-peptide-1 (GLP-1) analog of structure, its 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)
Wherein：
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. Africa xenopus glucagon-like-peptide-1 (GLP-1) analog according to claim 1, sequence preferably 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-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-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 kind of pharmaceutical composition, including the high blood of Africa xenopus pancreas described at least one claim 1 of therapeutically effective amount Sugared (GLP-1) analog of element sample peptide -1 and its pharmaceutically acceptable salt.
4. a kind of pharmaceutical composition, including the high blood of Africa xenopus pancreas described at least one claim 1 of therapeutically effective amount Sugared (GLP-1) analog of element sample peptide -1 and pharmaceutically acceptable carrier or diluent.
5. a kind of Africa xenopus glucagon-like-peptide-1 (GLP-1) analog as described in claim 3 and its pharmaceutically Acceptable salt is being prepared for the utilization in diabetes or the medicine of fat-reducing.
6. a kind of Africa xenopus glucagon-like-peptide-1 (GLP-1) analog as described in claim 4 and pharmaceutically may be used The carrier or diluent of receiving are being prepared for the utilization in diabetes or the medicine of fat-reducing.
7. a kind of preparation method of Africa xenopus glucagon-like-peptide-1 (GLP-1) analog as described in claim 1, Including biological expression, liquid phase synthesis and solid phase synthesis preparation method thereof.
CN201611156564.XA 2016-12-14 2016-12-14 A kind of Africa xenopus glucagon-like peptide 1（GLP‑1）Analog and its application Pending CN107266556A (en)

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CN112724240A (en) * 2017-11-24 2021-04-30 江苏师范大学 Xenopus laevis glucagon-like peptide-1 analogue and application thereof

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Application publication date: 20171020