阅读说明：本技术 不同构型的glp-1类似肽修饰二聚体及其制备方法在治疗ii型糖尿病中的应用 (GLP-1 analog peptide modified dimer with different configurations and application of preparation method thereof in treating type II diabetes ) 是由 唐松山 罗群 张旭东 董玉霞 唐婧晅 杨莉 李玉华 葛平 戴小敏 于 2019-11-20 设计创作，主要内容包括：本发明提供了不同构型新型胰高血糖素肽1脂肪酸修饰或非修饰二聚体在治疗II糖尿病中胰腺保护或降糖效果的应用。本发明的二聚体是两个相同含有半胱氨酸的GLP-1单体通过半胱氨酸氧化形成的二硫键连接而成。本发明的H型GLP-1同源二聚体(在肽链内部形成二硫键)在不降低活性情况下显著增加了GLP-1二聚体降糖持续时间,所提供的GLP-1类似物二聚体在体内持续活性长达19天,较阳性对照药利拉鲁肽体内活性为3天,或者和目前已经报道的长效GLP1类似肽比较,都有显著延长,极大地推动了长效GLP1类药物的技术进步和便利了其临床应用和推广。同时U型同源二聚体(在肽链C末端形成二硫键)不影响血糖,但可以明显保护胰腺腺泡和导管等外分泌部细胞,保护胰腺的功能。(The invention provides application of a novel glucagon peptide 1 fatty acid modified or unmodified dimer with different configurations in pancreatic protection or blood sugar reduction effect in treatment of diabetes II. The dimer is formed by connecting two identical GLP-1 monomers containing cysteine through a disulfide bond formed by oxidizing the cysteine. The H-type GLP-1 homodimer (disulfide bond is formed in the peptide chain) obviously prolongs the glucose-reducing duration time of the GLP-1 dimer under the condition of not reducing the activity, the provided GLP-1 analog dimer has the continuous activity in vivo for 19 days, and the activity in vivo is 3 days compared with that of a positive control drug liraglutide, or compared with long-acting GLP1 similar peptide reported at present, the GLP-1 analog dimer obviously prolongs the activity in vivo, and greatly promotes the technical progress of long-acting GLP1 drugs and facilitates the clinical application and popularization of the GLP-1 analog dimer. Meanwhile, the U-shaped homodimer (forming a disulfide bond at the C terminal of a peptide chain) does not influence blood sugar, but can obviously protect exocrine cells such as pancreatic acini, ducts and the like and protect the functions of pancreas.)

1. A monomeric glucagon-like peptide 1 analog peptide, wherein the amino acid sequence of said glucagon-like peptide 1 analog peptide is any one of the following four:

(1)

His-X₈-Glu-Gly-Thr-Phe-Thr-Cys-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-X₃₇(ii) a Or

(2)

His-X₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Cys-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-X₃₇(ii) a Or

(3)

His-X₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Cys-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-X₃₇(ii) a Or

(4)

His-X₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-Gly-Cys-OH；

Wherein, X₈Is L-alpha-alanine or β -alanine or α -aminoisobutyric acid or β -aminoisobutyric acid;

X₂₆lysine or lysine modified by glutamic acid alkyl on side chain epsilon amino or lysine modified by amino acid alkyl on side chain epsilon amino;

X₃₄is Arg or Lys or a lysine modified by the glutamic acid on the side chain epsilon amino;

X₃₅is Gly or Ala or β -alanine or alpha-aminoisobutyric acid or β -aminoisobutyric acid;

X₃₇is Gly-OH or Gly-NH₂Or NH₂Or an OH structure; or is allosteric, the amino acid sequence of the first 7-36 position of claim 1 consisting of 1 copy of a similar repeat sequence, position 8 (X) in the repeat sequence₈) Alanine by glycine or alpha-or β -aminoisobutyric acid (Aib), cysteine by serine or glycine, X in the repeat sequence₂₆Is arginine; or the C-terminal amido group is connected with a polyethylene glycol molecule to form a PEGylation modification, and the molecular weight of the PEG is 0.5-30KD。

2. The monomeric glucagon-like peptide 1 analog of claim 1, wherein when X is₂₆When the lysine is lysine modified by glutamyl [ gamma-Glu (N- α -alkanoic acid group) ] on side chain epsilon amino, the structural formula is shown in formula 1, or when the X is₂₆When the side chain epsilon amino is lysine modified by alkanoic acid group, the structural formula is shown as a formula 2; formula 1, 2 wherein n is 14 or 16;

3. a glucagon-like peptide 1 analog peptide homodimer, which is characterized in that the dimer is formed by connecting the same monomers of claims 1-2 through a disulfide bond formed by cysteine to form an H-type or U-type glucagon-like peptide 1 analog peptide homodimer, and the amino acid sequence of the dimer is any one of the following four:

wherein, X₈Is L-alpha-alanine (Ala) or β -alanine (β Ala) or α -or β -aminoisobutyric acid (alpha Aib or β Aib);

X₂₆lysine or lysine modified by glutamic acid alkyl on side chain epsilon amino or lysine modified by amino acid alkyl on side chain epsilon amino;

X₃₄is Arg or Lys or a lysine modified by the glutamic acid on the side chain epsilon amino;

X₃₅is Gly or Ala or β -alanine or alpha-aminoisobutyric acid or β -aminoisobutyric acid;

X₃₇is Gly-OH or Gly-NH₂Or NH₂Or an OH structure; or is changedThe amino acid sequence of positions 7 to 36 of claim 1, which is composed of 1 copy of a similar repeat sequence, position 8 (X) of the repeat sequence₈) Alanine by glycine or alpha-or β -aminoisobutyric acid (Aib), cysteine by serine or glycine, X in the repeat sequence₂₆Is arginine; or the C-terminal amido group is connected with a polyethylene glycol molecule to form a PEGylation modification, and the molecular weight of the PEG is 0.5-30 KD.

4. The glucagon-like peptide 1-like peptide dimer of claim 3, wherein when X is₂₆When the lysine is lysine modified by glutamyl [ gamma-Glu (N- α -alkanoic acid group) ] on side chain epsilon amino, the structural formula is shown in formula 1, or when the X is₂₆When the side chain epsilon amino is lysine modified by alkanoic acid group, the structural formula is shown as a formula 2; in the formulas 1 and 2, n is 14 or 16.

5. Use of a monomeric glucagon-like peptide 1-like peptide according to claims 1-2, or a GLP 1-like peptide dimer according to any one of claims 3-4, for the preparation of a pancreatic protective or/and hypoglycemic medicament for the treatment of diabetes mellitus type II.

6. A medicament for protecting pancreas or treating type II diabetes, which comprises a monomeric glucagon-like peptide 1 analog according to claims 1-2; or a glucagon-like peptide 1 analog peptide homodimer according to any one of claims 3 to 4 as active ingredient.

Technical Field

The invention belongs to the field of medical biology, and particularly relates to preparation of various novel human GLP1 analog peptide monomers or homodimers and application thereof in treating diabetes.

Background

Glucagon-like peptide 1(GLP 1) from the glucagon pro-glucagon protein is a 30 amino acid residue incretin-like peptide that is released by intestinal L cells upon nutrient intake, it enhances insulin secretion from pancreatic β cells, increases insulin expression and peripheral glucose utilization, inhibits β cell apoptosis, promotes satiety and β cell neogenesis, decreases glucagon secretion, and delays gastric emptying multiple effects make GLP1 receptor agonists of significant significance for the treatment of type 2 diabetes mellitus.

Exendin-4 is an incretin analogue isolated from saliva of Heloderma subspecium, has 39 amino acids and 53% sequence homology with GLP-1. Exenatide is an Exendin-4 synthetic molecule, has a long half-life period (3.3-4.0 hours) and a long-acting anti-hyperglycemic effect, and is administered twice a day.

Liraglutide is a GLP-1 analogue, and has 97% homology with natural human GLP-1. It contains Arg →³⁴Lys is substituted and is at²⁶Lys increases glutamyl palmitoyl chains. After subcutaneous injection, the final elimination half-life averages 13 hours, allowing once-a-day administration, whose pharmacokinetic properties are not affected by age, sex, kidney or liver function.

PB-105 is prepared by replacing cysteine at position 39 of Exenatide and performing specific pegylation modification on the cysteine, and PB-110(PEG5kd), PB-106(PEG20kd), PB-107(PEG30kd) and PB-108(PEG40kd) are prepared. The plasma T1/2 of PB-106 is about 10 times that of PB-105, and shows better hypoglycemic activity, but the hypoglycemic activity (specific activity) per milligram is reduced by more than 90%.

Lixisenatide is a novel long-acting GLP-1R agonist comprising 44 amino acids and is structurally similar to Exendin-4 except that there is no proline at position 38 and 6 lysine residues are added at position 39. In 24-week clinical medication, Lixisenatide was injected once daily and significantly reduced in activity, with a similar proportion of side effects of treatment in the Lixisenatide group as the control group (Lixisenatide 2.5% and placebo 1.9%), and a symptomatic hypoglycemia rate of (Lixisenatide 3.4% and placebo 1.2%).

BPI-3016 structurally modifies the bond (DIM) between positions 8 (Ala) and 8-9 (GLU) of human GLP-1.⁸The side chain of-CH 3 in Ala is replaced by-CF 3, the carbonyl group in the bond is converted to a methyl group, Lys → palmitoylation is performed²⁶Arg replaces and adds Gly at the C terminal. After single administration, the half-life of BPI-3016 on diabetic cynomolgus monkey exceeds 95 hours, FPG and postprandial blood sugar (PPG) are obviously reduced one week after administration, Body Mass Index (BMI) is reduced,Body fat, improved glucose tolerance, showing an insulin increasing effect.

Albiglutamide is a recombinant fusion protein, which is composed of two linked copies of human GLP-1 gene and human albumin gene in series. Gly →⁸Ala substitutions confer resistance to hydrolysis by DPP-4, allowing for once weekly dosing. Studies have shown that Albiglutide can reduce blood glucose parameters (HbA1c, PPG, and FPG), thereby enhancing glucose-dependent insulin secretion and slowing gastric emptying.

Dulaglutide is a GLP-1 analog fused to an Fc fragment with the structure of Gly⁸Glu²²Gly³⁶-GLP-1(7-37)-(Gly₄Ser)₃-Ala-Ala^234,235Pro²²⁸IgG 4-Fc. Dulaglutide is administered once a week. Dulaglutide showed a higher reduction in HbA1c compared to placebo, metformin, insulin glargine, sitagliptin and Exenatide. Dulaglutide has various therapeutic effects of reducing weight, reducing the progression of renal disease, reducing the incidence of myocardial infarction, reducing blood pressure and the like in the treatment of T2D.

Semaglutide is a GLP1 long-acting analogue peptide with Aib →⁸Ala substitutions and²⁶lys a longer linker (2 xAEEAC-delta-glutamyl- α -oleic diacid). it maintained 94% GLP1 homology compared to Liraglutide, Semaglutide activity decreased 3-fold but albumin binding increased, predicted to have a 165-fold 184 hour half-life (7 days). Semaglutide showed significant HbA1c and weight loss.

Taspoglutide contains α -aminoisobutyric acid Aib →⁸Ala and³⁵hGLP-1(7-36) NH of Gly₂. Taspoglutide has a strong affinity constant with GLP-1R and is completely resistant to aminodipeptidase. In a 24-week clinical study, Taspoglutide significantly reduced HbA1c, FPG and body weight. But the side effects are obvious.

GLP-1 analog studies still need to be optimized because current long-acting activators have proven less effective than Liraglutide or native GLP1 in terms of specific activity (hypoglycemic effect in mg), dosing dose, weight loss, and side effects, such as a weight loss of 0.6 kg for Albiglutide and 2.2 kg for Liraglutide, a weight loss of 2.9 kg for the Dulaglutide group and 3.6 kg for the Liraglutide group in a 26 week trial. In rodents, Semaglutide causes a dose-dependent and treatment duration-dependent thyroid C cell tumor. Clinical studies showed that 57.2% of patients with normal renal function, 35.9% of patients with mild impairment and 6.9% of patients with moderate impairment. Patients taking Semaglutide experienced a higher frequency of gastrointestinal adverse reactions such as nausea, vomiting, diarrhea, abdominal pain and constipation than placebo (15.3% for placebo, 32.7 and 36.4% for Semaglutide 0.5 and 1 mg). When the Semaglutide and the sulfonylurea medicines are used together, 0.8-1.2% of patients have severe hypoglycemia, injection site discomfort and erythema are 0.2%, the average amylase of the patients is increased by 13%, and the lipase is increased by 22%. The incidence rates of cholelithiasis are 1.5% and 0.4%, respectively.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a glucagon-like peptide 1 analog peptide monomer and a homodimer thereof.

The invention aims to provide a glucagon-like peptide 1 analog peptide monomer, wherein the amino acid sequence of the glucagon-like peptide 1 analog peptide is any one of the following four types:

(1)

His-X₈-Glu-Gly-Thr-Phe-Thr-Cys-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-X₃₇(ii) a Or

(2)

His-X₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Cys-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-X₃₇(ii) a Or

(3)

His-X₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Cys-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-X₃₇(ii) a Or

(4)

His-X₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-X₂₆-Glu-Phe-Ile-Ala-Trp-Leu-Val-X₃₄-X₃₅-Arg-Gly-Cys-OH；

Wherein, X₈Is L-alpha-alanine (Ala) or β -alanine (β Ala) or α -or β -aminoisobutyric acid (alpha or β Aib);

X₂₆lysine, glutamic acid modified lysine of side chain epsilon amino acid or lysine modified by amino acid of side chain epsilon amino acid;

X₃₄is Arg, Lys or lysine modified by glutamic acid as the side chain epsilon amino alkanoate;

X₃₅is Gly or Ala or β -alanine or alpha-aminoisobutyric acid or β -aminoisobutyric acid;

X₃₇is Gly-COOH (glycine carboxyl terminal) or Gly-NH₂(amidated end of glycine) or NH₂(amidated end of arginine at position 36) or OH (carboxy end of arginine at position 36); or the first 7-36 amino acid sequence for allosteric use as provided for the first object is made up of 1 copy of a similar repeat sequence, position 8 (X) in the repeat sequence₈) Alanine by glycine or alpha-or β -aminoisobutyric acid (Aib), cysteine by serine or glycine, X in the repeat sequence₂₆Is arginine; or the C-terminal amido group is connected with a polyethylene glycol molecule to form a PEGylation modification, and the molecular weight of the PEG is 0.5-30 KD.

Preferably, when said X is₂₆When the lysine is lysine modified by glutamyl [ gamma-Glu (N- α -alkanoic acid group) ] on side chain epsilon amino, the structural formula is shown as formula 1, and when the X is₂₆When the side chain epsilon amino is lysine modified by alkanoic acid group, the structural formula is shown as a formula 2; formula 1, 2 wherein n is 14 or 16:

the second purpose of the invention is to provide a glucagon-like peptide 1 analog peptide homodimer, wherein the dimer is formed by connecting two same monomers through a disulfide bond formed by cysteine to form H-type or U-type glucagon-like peptide 1 analog peptide homodimer.

Preferably, the amino acid sequence of the dimer is any one of the following four:

wherein, X₈Is L-alpha-alanine (Ala) or β -alanine (β Ala) or α -or β -aminoisobutyric acid (alpha or β Aib);

X₂₆lysine, glutamic acid modified lysine of side chain epsilon amino acid or lysine modified by amino acid of side chain epsilon amino acid;

X₃₄is Arg, Lys or lysine modified by glutamic acid as the side chain epsilon amino alkanoate;

X₃₅is Gly, or Ala, or β -alanine, or alpha, or β -aminoisobutyric acid (Aib);

X₃₇is Gly-COOH (glycine carboxyl terminal) or Gly-NH₂(amidated end of glycine) or NH₂(amidated end of arginine at position 36) or OH (carboxy end of arginine at position 36); or the first 7-36 amino acid sequence for allosteric use as provided for the first object is made up of 1 copy of a similar repeat sequence, position 8 (X) in the repeat sequence₈) Alanine by glycine or alpha-or β -aminoisobutyric acid (Aib), cysteine by serine or glycine, X in the repeat sequence₂₆Is arginine; or the C-terminal amido group is connected with a polyethylene glycol molecule to form a PEGylation modification, and the molecular weight of the PEG is 0.5-30 KD.

Preferably, when said X is₂₆When the lysine is lysine modified by glutamyl [ gamma-Glu (N- α -alkanoic acid group) ] on side chain epsilon amino, the structural formula is shown as formula 1, and when the X is₂₆When the lysine is lysine modified by alkanoic acid group on side chain epsilon amino, the structural formula is shown as formula 2, and n is 14 or 16 in formula 1 and formula 2.

The third purpose of the invention is to provide the application of the monomeric glucagon-like peptide 1 analog peptide or the dimeric GLP1 analog peptide in preparing pancreatic protection or/and hypoglycemic drugs for treating II diabetes.

The fourth purpose of the invention is to provide a medicament for protecting pancreas or treating II diabetes, which takes the monomer glucagon-like peptide 1 analog peptide or the dimer glucagon-like peptide 1 analog peptide as the active ingredient.

The invention has the following advantages: under the condition that the activity of the H-like GLP-1 analog homodimer is not reduced, the hypoglycemic action time of the protective monomer GLP-1 peptide is obviously prolonged by 2-4 times (namely the specific activity of the dimer peptide is obviously improved), and the GLP-1R activator medicament approved by FDA is obviously prolonged. The GLP-1 analogue homodimer has the activity maintenance time in vivo of 19 days at most, is obviously prolonged compared with the Liraglutide serving as a positive medicament, obviously promotes the technology upgrade, and greatly facilitates the clinical application and market popularization. The U-like dimer does not affect the blood sugar level, but obviously protects exocrine cells such as pancreatic acini, ducts and the like, protects the pancreatic function and can be used for treating pancreatic related diseases.

Drawings

FIG. 1 is a graph showing the results of a blood glucose test on a single OGTT.

FIG. 2 is a graph showing the body weight changes of 2G2-2G8 in multiple OGTT tests.

FIG. 3 is a graph of weight change for the treatment of T2D model with 2G 3.

FIG. 4 is a graph showing the effect of 2G3 on reducing blood glucose in the T2D model of treatment.

FIG. 5 is a graph showing the result of H-E staining of pancreatic tissue treated by T2D model.

Fig. 6 is a graphical representation of Ki67 protein expression in dimer 2G 3-treated T2D model.

Figure 7 is a graphical representation of Ki67 protein expression in dimer 2G1 treatment T2D model.

FIG. 8 is a graph showing the results of TUNEL staining analysis.

FIG. 9 is a graph showing the results of GLP-1R staining analysis.

FIG. 10 is a schematic diagram showing the results of Western blot analysis of GLP-1R.

FIG. 11 is a graph showing the results of the insulin staining analysis (A: insulin staining; B: insulin staining analysis; C: islet number analysis).

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.