CN113754752A - Polypeptide and preparation method and application thereof - Google Patents

Polypeptide and preparation method and application thereof Download PDF

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CN113754752A
CN113754752A CN202111026493.2A CN202111026493A CN113754752A CN 113754752 A CN113754752 A CN 113754752A CN 202111026493 A CN202111026493 A CN 202111026493A CN 113754752 A CN113754752 A CN 113754752A
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polypeptide
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ser
glu
ala
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CN113754752B (en
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曾能
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Fitch Biomedical Zhongshan Research Institute Co ltd
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    • C—CHEMISTRY; METALLURGY
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention discloses a polypeptide and a preparation method and application thereof. The amino acid sequence of the polypeptide is His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (P EG2-PEG 2-gamma-Glu-CO (CH)2)18CO2H)‑Glu‑Phe‑Ile‑Ala‑Trp‑Leu‑Val‑Arg‑Gly‑Arg‑Gly the polypeptide prepared by the scheme has better and excellent long-acting blood glucose control effect than Somaolu peptide, has more stable blood concentration, has the effects of high enzymolysis stability, high biological activity and no adverse reaction, and has important significance in preparing medicaments for treating obesity and diabetes caused by hyperglycemia.

Description

Polypeptide and preparation method and application thereof
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a polypeptide, and a preparation method and application thereof.
Background
GLP-1 (glucagon-like peptide-1) is an intestinal hormone in a human body, and after eating, the hormone can promote insulin secretion (insulin generated by the 'incretin effect' accounts for more than 50% of the total amount of the insulin after eating), plays a role in glucose concentration dependent blood glucose reduction, is very intelligent, and only acts when the blood glucose is high, so that the risk of hypoglycemia caused by injection type insulin is avoided. It is further desirable that GLP-1 has an effect of protecting pancreatic islet beta cells: promote the transcription of insulin gene and the synthesis and secretion of insulin, stimulate the proliferation and differentiation of beta cells of pancreatic islets, inhibit the apoptosis of the beta cells of pancreatic islets and increase the number of the beta cells of pancreatic islets. GLP-1 can also act on islet alpha cells to strongly inhibit the release of glucagon, and act on islet delta cells to promote the secretion of somatostatin, and somatostatin can also be used as a paracrine hormone to participate in inhibiting the secretion of glucagon.
Disadvantages of GLP-1: GLP-1 produced by a human body is very easy to be degraded by dipeptidyl peptidase IV (DPP-4) in the body, the half-life period of plasma is less than 2 minutes, and the therapeutic effect can be produced only by continuous intravenous drip or continuous subcutaneous injection, which greatly limits the clinical application of the GLP-1.
Somalutide is a potent GLP-1 receptor agonist. The somagluteptide has more than 30 amino acids, and its main structural characteristics include the substitution of Aib for amino acid at catalytic site of DPP-4 hydrolase8(2-Aminoisobutyric acid) at amino acid Lys26Octadecyl chain alkyl diacid is grafted on the slow release carrier through the spacer so as to combine with albumin and delay renal clearance. Although the long-acting properties of the somaglutide have been improved, the patient's pursuit for long-acting properties has not yet been met.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a polypeptide which can control sugar for a long time.
The invention also provides a preparation method of the polypeptide.
The invention also provides an application of the polypeptide.
According to one aspect of the present invention, there is provided a polypeptide having the amino acid sequence His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(PEG2-PEG2-γ-Glu-CO(CH2)18CO2H) Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, abbreviated: HsEGTFTSDVSSYLEGQAA-K (PEG)2-PEG2-γGlu-CO(CH2)18COOH) -EFIAWLVRGRG; the molecular weight is 4171.73.
In a second aspect of the present invention, there is provided a method for producing a polypeptide having the above structure, the method comprising the steps of: taking solid-phase synthetic resin as an initial raw material, and sequentially connecting amino acids by adopting an Fmoc/t-Bu solid-phase synthesis strategy to obtain a polypeptide main chain with an amino acid protecting group; grafting a side chain on a polypeptide main chain, and carrying out deprotection group and peptide cutting; the side chain is PEG2-PEG 2-gamma-Glu-CO (CH)2)18COOH; the amino acid connecting sequence of the main chain of the polypeptide is His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly.
In some embodiments of the invention, the position of the grafted side chain is at amino acid lysine at position 20 of the backbone.
In some embodiments of the invention, the Resin is a Wang Resin.
In some embodiments of the invention, sequentially linking the amino acids using the Fmoc/t-Bu solid phase synthesis strategy further comprises assembling an Fmoc-Gly-Wang Resin vector.
In some embodiments of the present invention, when assembling the Fmoc-Gly-Wang Resin carrier, the addition ratio of Fmoc-Gly-OH to Resin is (4-8): 1.
in some embodiments of the present invention, the obtaining of the polypeptide backbone further comprises the step of coupling amino acids with 1-Hydroxybenzotriazole (HOBT), N-Diisopropylcarbodiimide (DIC) as a coupling agent, and N, N-Dimethylformamide (DMF) as a solvent, and removing Fmoc protecting groups with a 10-30% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) solution.
In some embodiments of the invention, the Fmoc group removal step is to wash the resin with 10-30% piperidine/DMF (v/v) solution to remove the protecting group.
In some embodiments of the invention, the method further comprises the steps of detecting the coupled amino acid and removing the Fmoc group using ninhydrin.
In some embodiments of the invention, the ratio of amino acid addition to DIC and HOBt is 1 (1-2) to (1-3).
In some embodiments of the invention, the amino acid protecting group is one or more of pbf, Boc, Fmoc, tBu, OtBu, and Trt.
In some embodiments of the invention, a lysing solution is used to perform the simultaneous deprotection and cleavage steps.
In some embodiments of the present invention, the composition of the lysis solution comprises TFA (trifluoroacetic acid), Phenol (Phenol), thioanisole (thioanisole), and EDT (1, 2-ethanedithiol), and the mass ratio is (80-90): (3-6): (3-6): (2-5).
The third aspect of the invention provides an application of the polypeptide, wherein the application of the polypeptide is an application in preparing a medicament for treating or preventing diabetes.
In some embodiments of the invention, the use of the polypeptides in the preparation of glucagon-like peptide-1 receptor agonists
In some embodiments of the invention, the use of the polypeptide in the preparation of a glucagon receptor (GCGR) agonist.
In some embodiments of the invention, the polypeptide is used for preparing a medicament for treating or preventing obesity caused by hyperglycemia.
A medicament for treating diabetes comprises the polypeptide.
In some embodiments of the present invention, the raw materials for preparing the medicament for treating diabetes further comprise a pharmaceutically acceptable carrier.
In some embodiments of the invention, the pharmaceutically acceptable carrier is a pharmaceutical carrier that is conventional in the pharmaceutical arts.
In some embodiments of the invention, the pharmaceutically acceptable carrier comprises at least one of a diluent, excipient, filler, binder, disintegrant, absorption enhancer, surfactant, adsorptive carrier, lubricant, sweetener, and flavoring agent.
In some embodiments of the invention, the excipient comprises water.
In some embodiments of the invention, the bulking agent comprises at least one of starch and sucrose.
In some embodiments of the invention, the binding agent comprises at least one of a cellulose derivative, alginate, gelatin, and polyvinylpyrrolidone.
In some embodiments of the invention, the humectant comprises glycerin.
In some embodiments of the invention, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.
In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.
In some embodiments of the invention, the surfactant comprises cetyl alcohol.
In some embodiments of the invention, the adsorbent support comprises at least one of kaolin and bentonite.
In some embodiments of the invention, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.
In some embodiments of the present invention, the mass fraction of the polypeptide in the drug is 0.01% to 99%.
According to some preferred embodiments of the present invention, the polypeptide is present in the drug at a mass fraction of 0.05% to 95%.
According to some preferred embodiments of the present invention, the polypeptide is present in the pharmaceutical agent at a mass fraction of 0.05% to 20%.
In some embodiments of the invention, the diabetes drug is administered according to the following dose criteria: the polypeptide compound is 0.01 mg/day to 10 mg/day.
In some embodiments of the present invention, the dosage form of the drug is various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a soft capsule, a granule, a pill, an oral liquid, a dry suspension, a drop pill, a dry extract, an injection or infusion, a transdermal agent, or a transdermal microneedle.
In some embodiments of the present invention, the administration mode of the drug may be a conventional administration mode in the art, including but not limited to injection or oral administration. The injection can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
The term "administered dose" as used herein is an amount capable of alleviating or delaying the progression of a disease, degenerative or injurious condition. Depending on the particular disease being treated, as well as other factors including age, weight, health, severity of symptoms, route of administration, frequency of treatment, and whether other medications are concomitant during treatment.
According to the embodiment of the invention, at least the following beneficial effects are achieved:
1. obesity can induce endoplasmic reticulum stress, which impairs insulin signaling pathways by activating the serine phosphorylation levels of JNK and its downstream insulin receptors, liver lipid accumulation, and insulin resistance, which have been considered as a new important mechanism for insulin resistance in obesity. The polypeptide compound provided by the invention can weaken an insulin signal path and reduce AKT phosphorylation level and IRS-1 tyrosine phosphorylation level by replacing Aib aminoisobutyric acid with D-type serine in the second sequence amino acid. Inhibition of cytochrome P4504A expression reduces hepatic endoplasmic reticulum stress and insulin resistance. Endoplasmic reticulum stress plays an important role in insulin resistance, and can be used as a potential therapeutic target for diabetes. The innovative design ensures that the peptide chain has stronger activity, strong targeting property and more obvious effect of losing weight and controlling sugar.
2. The amino acid sequence of the scheme of the invention is a short peptide chain of a side arm connected with the 20 th Lys position in the amino acid sequence through the amino acid substitution of a specific position, namely: compared with the positive medicine of the somaglutide without the modification of the side arm, the polypeptide medicine designed by the invention greatly prolongs the action half-life period, realizes the ultra-long effect of the polypeptide medicine, and has the integral medicine action effect which is greatly superior to the positive medicine of the somaglutide by adopting the site-specific side arm modification technology. In addition, the polypeptide prepared by the invention utilizes lipophilic substituent groups to bind with albumin in blood, thereby protecting the albumin from enzyme degradation and improving half-life. The polypeptides prepared by the invention stabilize the helical structure of the molecule through intramolecular bridges, and improve the efficacy and/or selectivity of the polypeptides against glucagon-like peptide 1 receptor (GLP-1R). The prepared polypeptide has good stability, easy amplification production and low cost. Compared with the somaglutide, the long-acting property and the stability of the pharmaceutical effect are far better than those of the somaglutide (the half-life period is 2 times or more than that of the somaglutide).
3. The polypeptide prepared by the scheme has a better and excellent long-acting blood glucose control effect than the Somazole, the blood concentration is more stable, and the polypeptide has the effects of high enzymolysis stability, high biological activity and basically no adverse reaction, and has important significance in preparing obesity medicines and diabetes medicines caused by hyperglycemia.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a line graph showing the change of blood glucose in mice subjected to intragastric glucose administration 24 hours after administration of Uomargitide and Soomargitide in the test examples of the present invention;
FIG. 2 is a line graph showing the change of blood glucose in mice subjected to intragastric glucose administration 48 hours after administration of Uomargitide and Soomargitide in the test examples of the present invention;
FIG. 3 is a graph comparing the rate of change of blood glucose per minute in control and after administration of Umarumopeptide and Somarumopeptide in test examples of the present invention, wherein P is < 0.00001;
FIG. 4 is a graph showing the results of AUC of the effect of homo-and Somalupide on blood glucose in the test examples of the present invention, wherein P is < 0.005 and P is < 0.00001.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
EXAMPLE 1 preparation of the polypeptide
This example prepared a polypeptide, synthesized manually according to the Fmoc/t-Bu strategy, on a synthetic scale: 0.5mmol, the following polypeptides were synthesized: Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (PEG2-PEG 2-gamma-Glu-CO (CH 2)2)18COOH) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Wang Resin, the specific process is as follows:
1. master peptide chain Assembly
(1) 1.47g Wang Resin (loading 0.47mmol/g, Xian Zi) was weighed into a reaction column, N-Dimethylformamide (DMF) was added to swell for 30min, and Fmoc-Gly-OH: 1.233g (6eq), HOBT: 0.672g (7.2eq), DMAP: 0.06g (0.72eq) for use. The DMF was taken off, the resin was washed thoroughly with N, N-Dimethylformamide (DMF) 2 times, and the weighed material was added to the reaction column. An appropriate amount of DMF was added, nitrogen was added thereto and the mixture was stirred well, followed by addition of 0.83mL (7.8eq) of N, N-Diisopropylcarbodiimide (DIC). And (5) reacting for 2h, and finishing the reaction. The reaction was taken off, washed 3 times with DMF and blocked for 4h by addition of acetic anhydride/pyridine (7:6, v/v). And (4) pumping off the blocking solution, and washing with DMF for 6 times to obtain Fmoc-Gly-Wang Resin.
(2) Fmoc-Gly-Wang Resin is used as a carrier, 1-Hydroxybenzotriazole (HOBT) and N, N-Diisopropylcarbodiimide (DIC) are used as coupling agents, N, N-Dimethylformamide (DMF) is used as a solvent, an Fmoc group is removed by 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) solution (5 min +7min twice), and the deprotection and coupling effects are monitored by ninhydrin in the Fmoc removal and coupling processes. By manual feeding, Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Alloc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Tyr-OH, Fmoc-Leu-OH, (tBu) -OH, Fmoc-Ser (Fmoc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-D-Ser (t-Bu) -OH, Boc-His (Trt) -OH the above amino acid feeds (5 eq relative to the synthesis scale) to give Boc-His-D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser tBu-Asp (OtBu) -Val-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Glu (AltBu) -Ala-Ala-Boc-Ala-Ala-Ala-Ser (Tan) -Leu-Val-Arg (pbf) -Gly-Wang Resin. The reaction was completed and washed 6 times with N, N-Dimethylformamide (DMF) and 6 times with Dichloromethane (DCM).
(3) Removal of allyloxycarbonyl (Alloc) groups and introduction of lipophilic substituents
Washing the resin twice with a solution of N, N-Dimethylformamide (DMF)/Dichloromethane (DCM) ═ 1:1 (volume ratio), adding a solution of tetrakis triphenylphosphine palladium (Pd (PPh3)4) in DCM and morpholine, shaking the reaction mixture at room temperature for 2 hours, and then filtering to obtain Boc-his (Boc) -D-Ser (t-Bu) -glu (otbu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -asp (otbu) -Val-Ser (t-Bu) -Tyr (t-Bu) -Leu-glu (otbu) -Gly-Gln-Ala-Lys-glu (otbu) -Phe-Ile-Ala-trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) ) -Gly-WangResin. After this time the resin was washed thoroughly three times each with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
FmocNH-PEG2-OH (CAS #:872679-70-4, Quanta BioDesign) was added in an amount corresponding to 5 times of the synthetic scale, 1-Hydroxybenzotriazole (HOBT), N, N-Diisopropylcarbodiimide (DIC) were used as coupling agents in an amount of 1.2 times and 1.3 times the amount of amino acid charged, and the mixture was shaken for 2 hours and then filtered. Obtaining Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Ala-Lys (Fmoc-PEG2) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-WangResin). After this time the resin was washed thoroughly three times each with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
Removing Fmoc group from 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) (twice 5min +7min), adding FmocNH-PEG2-OH (CAS #:872679-70-4, Quanta BioDesign) corresponding to 5 times of synthetic scale, using 1-Hydroxybenzotriazole (HOBT) and N, N-diisopropyl carbodiimide (DIC) as coupling agent in an amount of 1.2 times and 1.3 times of the amount of amino acid respectively, shaking for 2 hours, and filtering to obtain (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Boc) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (t-Bu) -Gly-Gln-Ala) -Lys (Fmoc-PEG2-PEG2) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-WangResin. After this time the resin was washed thoroughly three times each with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
Removing Fmoc group from 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) (twice 5min +7min), adding Fmoc-Glu-OtBu corresponding to 5 times of synthetic scale, using 1-Hydroxybenzotriazole (HOBT), N, N-diisopropyl carbodiimide (DIC) as coupling agent in an amount of 1.2 times and 1.3 times of the amino acid charge respectively, shaking for 2 hours, and filtering to obtain Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Ala-Lys (Fmoc-PEG 2-gamma-2-gamma-D-N-Dimethylformamide (DMF) -Glu) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Wang Resin. After this time the resin was washed thoroughly three times each with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
Removing Fmoc group by 20 percent Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) (twice for 5min +7min), adding eicosanoic acid mono-tert-butyl ester which is 5 times of the synthetic scale, using 1-Hydroxybenzotriazole (HOBT) and N, N-diisopropyl carbodiimide (DIC) as coupling agents with the dosage of 1.2 times and 1.3 times of the dosage of amino acid respectively, after shaking for 2 hours,filtering to obtain Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Ala-Lys (PEG2-PEG 2-gamma-Glu-CO (CH 3578-gamma-Glu-CO (CH)2)18COOH) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Wang Resin. After this time the resin was washed thoroughly three times with N, N-Dimethylformamide (DMF), Dichloromethane (DCM) and Dichloromethane (DCM) in succession and dried in vacuo.
2. Removal of full protection of polypeptides
To dry Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Ala-Lys (PEG2-PEG 2-gamma-Glu-CO (CH 3578)2)18COOH) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Wang Resin, adding lysate TFA/Phenol/thioanisole/EDT/H2O (82.5: 5: 5: 2.5: 5, vol.) was reacted at room temperature for 2.5 hours in a 10-fold volume by weight. Filtering, washing the filter cake with a small amount of lysate for 3 times, and combining the filtrates. The filtrate was slowly poured into ice-cold ether with stirring. Standing for more than 0.5 hr, centrifuging to obtain precipitate, washing with glacial ethyl ether for 3 times, and vacuum drying to obtain crude product H-His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (PEG2-PEG 2-gamma-Glu-CO (CH)2)18COOH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
3. Purification of polypeptide Compound
Dissolving the crude product obtained in the above step in Acetonitrile (ACN)/H2In a solution of O ═ 1:2 (by volume), preparative HPLC purification was performed by a 10 μm reverse phase C18 preparative column (20mm × 250 mm). With 30% acetonitrile (containing 0.05% trifluoroacetic acid)/H2Starting with O (containing 0.05% trifluoroacetic acid), the column was eluted with a gradient (increasing the proportion of acetonitrile at a rate of 1.33%/min) at a flow rate of 15mL/min for 30 minutes, the fractions containing the peptide were collected and lyophilized to give a pure polypeptide with an HPLC purity of greater than 95%, which was designated as Youmaglutide (if purity was not satisfactory, one-time HPLC purification was repeated). Separated by LC-MS analysisThe product of (4), the product purity is greater than 95%.
Test examples
1. Determination of agonistic Activity of Umaruzide on GLP-1R and GCGR
This experimental example tested the agonistic activity of homo-and somaglutide prepared in the examples on GLP-1R and GCGR.
Experimental materials: somaloutide was purchased from Zhejiang surge peptide biology, Inc. (CAS No.: 910463-68-2). And performing primary screening on the agonistic activity of GLP-1R and GCGR on the Umaruzide in GLP-1R-Luciferase-293T cell models and GCGR-Luciferase-293T cell models.
The experimental method comprises the following steps: the digested cells were plated in 96-well plates (medium containing 10% FBS, GLP-1R-Luciferase-293T: 20000cell/well, GCGR-Luciferase-293T: 20000cell/well, 100. mu.L); after 36h, removing the culture medium in the 96-well plate, and adding 90 mu L of serum-free culture medium; after 6h, preparing a series of concentrations (0.01, 0.1, 1, 10, 100, 1000, 10000, 100000, 1000000, 10000000 and 100000000pM) of the umreuptadine and the somareuptadine by using a serum-free culture medium, adding 10 mu L (namely diluting by 10 times) of the umreuptadine and the somareuptadine into each hole, and incubating the cells for 5 h; adding 100 mu L of cell lysate into each well, cracking on ice for 10min, then shaking uniformly, taking 2 mu L of lysate, adding into a white board of a 384 enzyme-labeling instrument, adding 10 mu L of firefly luciferase reaction solution, reading, adding 10 mu L of renilla luciferase reaction solution, and reading; data processing, the number of the firefly luciferase reading is divided by the Renilla luciferase reading, and the blank value is subtracted to obtain the activation times at different concentrations.
The experimental results are as follows: EC of Somatolutide GLP-150EC for 265.2pM and GCGR508600875 pM; EC of Umarlutide polypeptide compound GLP-1 of the invention50EC of 315.2pM and GCGR5026008755 pM. The result shows that the excellent marreu peptide polypeptide compound prepared by the scheme of the application has more obvious agonistic activity on GLP-1R and GCGR as well as on somareu peptide, and has certain specificity on the activation of GLP-1R receptors.
2. Effect of Youmareuptate and Soumareuptate on oral glucose tolerance (OGTT)
The experimental method comprises the following steps: is aged for 8 weeksMale C57BL/6J mice (university of tokyo model animal research center) were randomly grouped into groups of 8 per group according to similar blood glucose (assessed by blood samples taken from the tip of the tail). After fasting (6 hours), animals were administered with the optimized marreuptade polypeptide compound of the present invention and the positive control doxomareuptade both subcutaneously at a dose of 100ug/kg, and PBS was administered to the control group. ddH for intragastric glucose2O is prepared into stock solution with the concentration of 0.5g/mL and is stored at normal temperature. The mice are fasted for 8h before gastric lavage, and are administered with gastric glucose at 2.5g/kg dose for 24h and 48h, respectively, and the gastric glucose is administered with ddH2O is prepared into stock solution with the concentration of 0.5g/mL and is stored at normal temperature. Mice were fasted for 8h before gavage, gavage with glucose at a dose of 2.5g/kg at 24h and 48h respectively, and blood glucose was measured at t 0min, t 15 min, t 30min, t 60 min, t 90 min and t 120min respectively. Blood glucose changes were measured and followed up to 48 hours. The data were processed using software GraphPadPrism to generate a line graph of blood glucose changes (as shown in figures 1 and 2) and the area under the curve was calculated to generate an AUC plot (as shown in figures 3 and 4).
The experimental results are as follows: the results are shown in fig. 1-4, fig. 1 is a line graph showing the change of blood glucose of mice when gavage glucose is performed on the mice 24h after administration of the homo-and somaglutide; FIG. 2 is a line graph showing the change of glucose in the blood sugar of mice when they are gavaged with homo-and somaglutide for 48 h; FIG. 3 is a graph comparing the rate of change of blood glucose per minute for control and administered prime and somaglutide; FIG. 4 is a graph of AUC results for the effect of homo-and somaglutide on blood glucose; as can be seen from the figure, both somalobulin and ummaritide had significantly lower AUC (P <0.05) at 24h after administration (0-120min) compared to vehicle (PBS) and started to increase to different extents at 48h following the OGTT curve. At AUC under the OGTT curve period of 48h, somaglutide had lost pharmacodynamic activity.
In conclusion, the activity of the umulptin prepared by the scheme of the invention is equivalent to that of the somaglutide, but the effect of the umulptin on long-acting blood sugar reduction is obviously better than that of the somaglutide. Compared with a carrier (PBS), the excellent marlutide polypeptide compound can improve the glucose tolerance of mice, and shows more excellent and remarkable glucose tolerance improving effect, and the result also shows that the excellent marlutide polypeptide compound has more excellent and remarkable long-acting glucose reducing effect.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims (10)

1. A polypeptide having an amino acid sequence of His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (P EG2-PEG 2-gamma-Glu-CO (CH)2)18CO2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
2. A method for producing the polypeptide of claim 1, comprising the steps of: taking solid-phase synthetic resin as an initial raw material, and adopting an Fmoc/t-Bu solid-phase synthesis strategy to sequentially connect and protect amino acids or polypeptides to obtain a polypeptide main chain with amino acid protecting groups; grafting side chain to the main polypeptide chain, deprotection, and cutting peptide.
3. The use of a polypeptide according to claim 1 for the preparation of a glucagon-like peptide-1 receptor agonist.
4. Use of the polypeptide of claim 1 in the preparation of a glucagon receptor agonist.
5. Use of the polypeptide of claim 1 for the preparation of a medicament for the treatment or prevention of hyperglycemia-induced obesity.
6. Use of the polypeptide of claim 1 for the preparation of a medicament for the treatment or prevention of diabetes.
7. A medicament for the treatment of diabetes comprising the polypeptide of claim 1.
8. The medicament for treating diabetes according to claim 7, wherein the raw materials for preparing the medicament further comprise a pharmaceutically acceptable carrier.
9. The medicament for treating diabetes according to claim 7, wherein the mass fraction of the polypeptide in the medicament is 0.01-99%; preferably, the mass fraction is 0.05% -95%; more preferably, the mass fraction is 0.05% to 20%.
10. The drug for treating diabetes according to claim 7, wherein the drug is administered in an amount standard of: polypeptide 0.01 mg/day-10 mg/day.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104302772A (en) * 2012-05-18 2015-01-21 爱德迪安（北京）生物技术有限公司 Protein and protein conjugate for diabetes treatment, and applications thereof
CN107129538A (en) * 2010-04-27 2017-09-05 西兰制药公司 Peptide conjugate of the receptor stimulating agents of GLP 1 and gastrin and application thereof
CN110922470A (en) * 2019-12-26 2020-03-27 杭州肽佳生物科技有限公司 Preparation method of somaglutide
CN112552391A (en) * 2019-09-25 2021-03-26 北京志道生物科技有限公司 Recombinant interleukin-15 analogue

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107129538A (en) * 2010-04-27 2017-09-05 西兰制药公司 Peptide conjugate of the receptor stimulating agents of GLP 1 and gastrin and application thereof
CN104302772A (en) * 2012-05-18 2015-01-21 爱德迪安（北京）生物技术有限公司 Protein and protein conjugate for diabetes treatment, and applications thereof
CN112552391A (en) * 2019-09-25 2021-03-26 北京志道生物科技有限公司 Recombinant interleukin-15 analogue
CN110922470A (en) * 2019-12-26 2020-03-27 杭州肽佳生物科技有限公司 Preparation method of somaglutide

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