CN113493504B - Molecular modification of GIP-Exendin-4 chimeric peptide and application of dimer thereof in treatment of diabetes - Google Patents

Abstract

The present invention provides glucose-dependent insulinotrophic polypeptide (GIP)-Exendin 4 chimeric polypeptide molecular isomers and dimers based on glucagon-like peptide-1 receptor activator (GLP-1R) in reducing blood sugar. and applications in the treatment of metabolic syndrome. The dimer of the present invention is formed by connecting two identical GIP-Exendin-4 chimeric peptide monomers containing a single allosteric cysteine through a disulfide bond formed by cysteine. The H-type dimer of the present invention (single Ser → Cys substitution in the molecule) significantly increases the hypoglycemic duration of the dimer without reducing the activity. The dimer of the present invention continues to be active in vivo for up to 22 days, which is significantly longer than the positive control drug Lixisenatide (2 days). Compared with Lixisenatide, dimer 2G21 at the same molar concentration produced similar hypoglycemic activity and weight loss, induced doubled insulin secretion, and improved organ toxicity, expanding the medical use of dimers, such as in the treatment of metabolic syndrome. , including uses in the treatment of diabetes and obesity, and in reducing excessive food intake.

Description

Molecular modification of GIP-Exendin-4 chimeric peptide and its dimer in the treatment of diabetes Applications in

Technical field

The invention belongs to the field of medical biology, and specifically relates to the molecular modification of GLP-1R activator-like peptide and the application of its homodimer in the treatment of metabolic diseases.

Background technique

Exendin-4 is an incretin analog isolated from the saliva of Heloderma suspectum. It has 39 amino acids and has 53% sequence homology with GLP-1. GIP is a 42-amino acid gastrointestinal regulatory peptide, which has the function of regulating the body's glucose metabolism, promoting the release of insulin from pancreatic beta cells, and reducing body weight. GLP-1 is a 30-amino acid residue incretin-like peptide that is released from intestinal L cells upon nutrient intake. Exendin-4 and GLP-1 are the two GLP-1R activators discovered so far. Based on the amino acid sequences of these three active polypeptides that regulate glucose metabolism, after nearly ten years of significant structural changes, each allosteric birth of a hypoglycemic GLP-1R activator that has been marketed or clinically approved by the US FDA or China SFDA, such as each Once-daily dosing Liraglutide (launched in 2011) and Lixisenatide (launched in 2016), twice-daily dosing Exenatide (launched in 2010) and once-weekly dosing Polyethylene Clycol Loxenatide (launched in 2019), Albiglutide (launched in 2014) ), Dulaglutide (launched in 2014), Semaglutide (launched in 2017), Taspoglutide (clinical failure in 2015) and Tirzepatide (Phase II clinical trial in 2018). Exenatide, Polyethylene Clycol Loxenatide and Lixisenatide are allosteric molecules based on the amino acid sequence of the active polypeptide Exendin-4, and they have completed clinical entry into the market. Liraglutide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide are allosteric analogs based on the amino acid sequence of the active polypeptide GLP-1. They are produced through chemical synthesis or genetic recombination-chemical synthesis combined methods. Tirzepatide is a synthetic molecule based on the GIP-Exendin-4 bifunctional receptor activator. It was developed by Lily and has completed Phase II clinical trials.

Nausea and vomiting may occur during treatment with most GLP-1R activators. Since the hypothalamic–pituitary–adrenal axis (HPA or HTPA) is part of the physiological stress response, GLP-1R activators stimulate the HPA axis to increase corticosterone, leading to some heart rate abnormalities. Therefore, it is still necessary to: ① antagonize the activation of glucagon and GLP-2 receptors at the same time when activating GIP receptors and/or GLP-1 receptors; ② it is necessary to activate GIP receptors and/or GLP-1 receptors. body effects to provide weight loss, antagonize DPP-4 and other forms of degradation mechanisms, while maintaining low immunogenicity; ③GLP-1R activators still need to be optimized, because the current long-acting activators have low specific activity (unit It has been proven that they are not as effective as natural GLP-1 or Exendin-4 molecules in terms of quality of hypoglycemic effect), dosage, weight loss and side effects. At the same time, scientific and technological progress is needed to provide longer-acting hypoglycemic activators and overcome toxicity.

Contents of the invention

The object of the present invention is to provide a GLP-1R activator-like peptide. The GLP-1R activator-like peptide of the present invention performs molecular allostery on Exendin-4, GIP-Exendin-4 and GLP-1 respectively. On the one hand, the position of cysteine in the peptide chain was studied by replacing it with a single Cys→Ser inside the monomeric peptide. On the other hand, modification of fatty acids or fatty acid substituents on the ε-amino group of the lysine (K) side chain in the peptide chain is allosteric. Different fatty acids or fatty acid substituents have different effects on the activity of GLP-1R activator-like peptides. . The present invention also provides homodimers formed by the aforementioned GLP-1R activator-like peptides, and it is found that dimers with an H-type structure formed by cysteine residues at different positions (single Cys→Ser substitution in the molecule) produce different activities. , the sustained hypoglycemic time can be up to 22 days, which is significantly longer than the current clinical drugs that are once every 1-7 days.

In order to achieve the above object, the technical solution adopted by the present invention is: a glucagon-like peptide-1 receptor (GLP-1R) activator-like peptide, and the GLP-1R activator-like peptide is Exendin-4, GIP -The sequence of Exendin-4 chimeric peptide or GLP-1 is allosteric and modified with a fatty acid chain on the ε-amino group of lysine. The GLP-1R activator-like peptide of the present invention is based on the amino acid sequence of Exendin-4, GIP-Exendin-4 or GLP-1 peptide as the main chain, replacing Ser on the main chain with Cys, and in the main chain amino acid sequence A fatty acid chain containing only one Cys; the side chain is the ε-amino group of one of the lysines in the main chain.

Preferably, the specific sequence of the GLP-1R activator-like peptide is any one of the following:

(1)(HN ₂ )HX ₂ -EGTFTCDLS-X ₁₂ -QMEEEAV-X ₂₀ -LFIEWL-X ₂₇ -NGGPSSGAPP-X ₃₈ or;

(2)(HN ₂ )HX ₂ -EGTFTSDLC-X ₁₂ -QMEEEAV-X ₂₀ -LFIEWL-X ₂₇ -NGGPSSGAPP-X ₃₈ or;

(3)(HN ₂ )HX ₂ -EGTFTSDLS-X ₁₂ -QMEEEAV-X ₂₀ -LFIEWL-X ₂₇ -NGGPCSGAPP-X ₃₈ or;

(4)(HN ₂ )HX ₂ -EGTFTSDLS-X ₁₂ -QMEEEAV-X ₂₀ -LFIEWL-X ₂₇ -NGGPSCGAPP-X ₃₈ or;

(5)(HN ₂ )YX ₂ -EGTFTCDYSI-X ₁₃ -LDKIAQ-X ₂₀ -AFVQWLIAGGPSSGAPP-X ₃₈ or;

(6)(HN ₂ )YX ₂ -EGTFTSDYCI-X ₁₃ -LDKIAQ-X ₂₀ -AFVQWLIAGGPSSGAPP-X ₃₈ or;

(7)(HN ₂ )YX ₂ -EGTFTSDYSI-X ₁₃ -LDKIAQ-X ₂₀ -AFVQWLIAGGPCSGAPP-X ₃₈ or;

(8)(HN ₂ )YX ₂ -EGTFTSDYSI-X ₁₃ -LDKIAQ-X ₂₀ -AFVQWLIAGGPSCGAPP-X ₃₈ or;

(9)(HN ₂ )HX ₂ -EGTFTSDVSCYLEGQAA-X ₂₀ -EFIAWLV-X ₂₈ -GRG(NH ₂ );

Among them, X _{2 or} X _{13 is} L-α-glycine or L- _α -alanine or _α -aminoisobutyric acid (αAib); amino acid, or lysine modified with glutamyl fatty acid [γ-Glu(N-α-fatty acid)] or glutamyl fatty diacid [γ-Glu(N-α-fatty diacid)] on the side chain ε-amino group acid, or [2×AEEAC-γ-Glu-(N-α-fatty diacid)]-modified lysine on the ε-amino group of the side chain; X ₃₈ is PS (HN ₂ ) or SKKKKKK (HN ₂ ). The uppercase single letter is the abbreviation of L-ɑ-amino acid or the amino acid substitution symbol, the Arabic numerals are the order of amino acid residues, and NH ₂ represents the N-terminal or C-terminal amide group structure.

Preferably, the GLP-1R activator is similar to a peptide, when X ₁₂ or X ₂₀ or X _{27 or} When lysine is modified with aminoacyl fatty diacid [γ-Glu (N-α-fatty diacid)], its structure is shown in Chemical Formula 1; when X ₁₂ or X ₂₀ or X ₂₇ or X ₂₈ is the side chain ε- When lysine is modified with [2×AEEAC-γ-Glu-(N-α-fatty diacid)] on the amino group, its structure is shown in Chemical Formula 2.

The present invention also provides a hypoglycemic peptide homodimer, the dimer is connected by a disulfide bond formed between the same monomers according to any one of claims 1 to 3 through cysteine. into, forming an H-type GLP-1R activator-like peptide homodimer.

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

Among them, X _{2 or} X _{13 is} L-α-glycine or L- _α -alanine or _α -aminoisobutyric acid (αAib); amino acid, or lysine modified with glutamyl fatty acid [γ-Glu(N-α-fatty acid)] or glutamyl fatty diacid [γ-Glu(N-α-fatty diacid)] on the side chain ε-amino group acid, or [2×AEEAC-γ-Glu-(N-α-fatty diacid)] modified lysine on the ε-amino group of the side chain; X ₃₈ is PS (HN ₂ ) or SKKKKKK (HN ₂ ); "|" represents a disulfide bond formed between two cysteines.

Preferably, when X ₁₂ or X ₂₀ or X _{27 or} -α-fatty diacid)] modified lysine, its structure is shown in Chemical Formula 1; when X ₁₂ or X ₂₀ or X ₂₇ or X ₂₈ is the side chain ε-amino group [2×AEEAC-γ-Glu- When lysine is modified with (N-α-fatty diacid), its structure is shown in Chemical Formula 2.

The present invention also provides the use of the GLP-1R activator-like peptide or the homodimer in the preparation of drugs for treating metabolic syndrome. Preferably, the metabolic syndrome conditions include hyperglycemia, diabetes and obesity.

The present invention also provides a drug for treating metabolic syndrome, which uses the GLP-1R activator-like peptide or homodimer as described above and its pharmaceutically acceptable salts as active ingredients.

Beneficial effects of the present invention: The H-type GLP-1R activator analogue homodimer of the present invention significantly increases the corresponding monomer activator or is approved by the FDA or SFDA when the glucose-lowering intensity is not lower than that of the corresponding monomeric peptide. The approved GLP-1R activator clinical drugs have a hypoglycemic effect about 2-3 times longer. The provided GLP-1R activator analogue homodimer maintains its activity in the body for up to 22 days, which is longer than that of positive drugs. Lixinaglutide (the effect lasts for 2 days) is significantly prolonged. Compared with clinical GLP-1R activators, the structural changes of the new dimer are very obvious, which greatly facilitates its clinical application and marketing.

Description of drawings

Figure 1 is a schematic diagram of the blood glucose test results of a single OGTT.

Figure 2 is a statistical analysis chart of body weight when 2G21 is used to treat T2D models.

Figure 3 is a statistical analysis chart of blood sugar in the T2D model treated with 2G21.

Figure 4 is a statistical analysis chart of glycated hemoglobin in the T2D model treated with 2G21.

Figure 5 is a statistical analysis chart of insulin in the T2D model treated with 2G21.

Figure 6 is a statistical analysis chart of alanine aminotransferase in the T2D model treated with 2G21.

Figure 7 is a statistical analysis chart of pancreatic amylase in the T2D model treated with 2G21.

Detailed ways

In order to demonstrate the technical solution, purpose and advantages of the present invention more concisely and clearly, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

Example 1 Preparation of monomeric peptides and dimers

1. Solid-phase chemical synthesis process of monomeric peptides: manual solid-phase peptide synthesis operation steps.

1. Resin swelling: Put the amino resin (amino resin for C-terminal amidation sequence) (purchased from Tianjin Nankai Synthetic Technology Co., Ltd.) into the reaction pot, and add 15 ml/ml of dichloromethane (DCM, Dikma Technologies Inc.) g resin, shake for 30 min. SYMPHONY 12-channel peptide synthesizer (SYMPHONY model, software Version.201, ProteinTechnologies Inc.).

2. Connect the first amino acid: remove the solvent through sand core suction filtration, add 3 times the mole of the first C-terminal Fmoc-amino acid (all Fmoc-amino acids are provided by Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), and then add 10 Double the molar amount of 4-dimethylaminopyridine (DMAP) and N,N'-dicyclohexylcarbodiimide (DCC), and finally add dimethylformamide (DMF) (purchased from Dikma Technologies Inc.) to dissolve, Shake for 30 minutes. Blocked with acetic anhydride.

3. Deprotection: Remove DMF, add 20% piperidine-DMF solution (15ml/g) for 5 minutes, filter to remove the solvent, and add 20% piperidine-DMF solution (15ml/g) for 15 minutes. Piperidine was provided by Sinopharm Shanghai Chemical Reagent Company.

4. Test: Remove the solvent. Take a dozen resin particles, wash them three times with ethanol, add one drop each of ninhydrin, KCN and phenol solution, and heat at 105-110°C for 5 minutes. If it turns dark blue, it is a positive reaction.

5. Wash the resin: wash twice with DMF (10ml/g), twice with methanol (10ml/g), and twice with DMF (10ml/g).

6. Condensation: Depending on the specific synthesis conditions, the following methods can be used alone or in combination in peptide synthesis:

Method a: three times the amount of protected amino acids and three times the amount of 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (HBTU, Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), Dissolve them with as little DMF as possible and add them to the reaction pot. Immediately add ten times the amount of N-methylmorpholine (NMM, Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.). The reaction lasted for 30 minutes and the test was negative.

Method b: Dissolve three times the amount of protected amino acid FMOC-amino acid and three times the amount 1-hydroxybenzotriazole (HOBt, Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.) with as little DMF as possible, add it to the reaction tube, and add it immediately Three times the amount of N,N'-diisopropylcarbodiimide (DIC) was reacted for 30 minutes, and the test was negative.

7. Wash the resin: wash once with DMF (10ml/g), twice with methanol (10ml/g), and twice with DMF (10ml/g).

8. Repeat steps 2 to 6. As shown in Table 1 for GLP-1R activating peptides with no side chain modification of amino acids, or GLP-1R activating peptides with side chain modifications, connect the corresponding amino acids in sequence from right to left. With K ₁₂ or K ₂₀ or K ₂₇ or K ₂₈ modification, synthesize it according to the following method 9.

9. Synthesis of K{N-ε-[γ-Glu-(N-α-fatty acid or fatty diacid)]}: Add 10ml of 2% hydrazine hydrate and react for 30 minutes to remove the protective group Dde of Fmoc-Lys(Dde)-OH. The side chain amino groups were exposed, washed six times with DMF and methanol alternately, and ninhydrin was detected as blue. Weigh 550 mg of Fmoc-Glu-OTBU and 250 mg of HOBT, dissolve in DMF, add 0.3 ml of DIC, mix well, add to the reactor to react with lysine side chain amino group for 1 hour, drain, wash with DMF 4 times, indene Triketone detects colorless. Add 5 ml of 20% piperidine DMF solution to the reactor and react for 20 minutes to remove the amino protection group Fmoc of Fmoc-Glu-OTBU. Wash alternately with DMF and methanol six times. Ninhydrin is detected as blue. Weigh 300 mg of fatty acids or fatty diacids and 250 mg of HOBT, dissolve them in DMF, add 0.3 ml of DIC, mix well, add them to the reactor to react for 1 hour, drain, and wash 4 times with DMF. Ninhydrin is colorless when detected. Wash with methanol twice and drain.

Synthesis of K{N-ε-[2×AEEAC-γ-Glu-(N-α-fatty diacid)]}: After removing the fmoc group from Dde-Lys(fmoc), add 2mM Fmoc-AEEAC-OH and 2mM benzotriazole hexafluorophosphate-1-oxytripyrrolidinyl phosphorus (PyBOP), 45mM HOBt, dissolve in DMF, add 0.375mM N,N'-diisopropylethylamine (DIPEA) in an ice water bath ) was activated for 3 minutes, added to the reaction column and reacted for 2 hours, and the end point of the experiment was determined by the ninhydrin method. At the end of the reaction, Fmoc was removed with 20% piperidine-DMF solution (15 ml/g) and washed with DMF 6 times. Fmoc-AEEAC-OH, Fmoc-Glu-OtBu and fatty diacid chain groups were coupled again in the same way. React with 2% hydrazine hydrate for 30 minutes to remove the sequence lysine protecting group Dde, and then attach it to the ε amino group of the lysyl side chain through step 8.

10. Pass the condensed polypeptide through DMF (10ml/g) twice, DCM (10ml/g) twice, DMF (10ml/g) twice, and drain for 10 minutes. Ninhydrin test was negative.

11. Remove the FMOC protecting group of the last N-terminal amino acid of the peptide chain. If the test is positive, drain the solution and set aside.

12. Wash the resin according to the following method, followed by DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice, DCM (10ml/g) twice, and drain it for 10 minutes.

13. Cleave peptides from resin: Prepare cutting solution (10 ml/g): TFA 94% (J.T. Baker Chemical Company), water 2.5%, ethanedithiol (EDT, Sigma-Aldrich Chemistry) 2.5% and triisopropylsilane (TIS, Sigma-Aldrich Chemistry) )1%. Cutting time: 120min.

14. Blow dry and wash: Blow the lysate as dry as possible with nitrogen, wash six times with ether, and then evaporate to dryness at room temperature.

15. Use the following method to HPLC purify the peptide, identify it and store it at -20°C in the dark.

2. The inspection methods are as follows:

1. Use HPLC to purify polypeptides: Dissolve the crude peptide in pure water or add a small amount of acetonitrile, and purify according to the following conditions: high performance liquid chromatography (analytical type; software Class-VP.Sevial System; manufacturer Japan SHIMADZU) and Venusi MRC-ODS C18 chromatographic column (30×250mm, Tianjin Bonna-Agela Technologies). Mobile phase A solution: 0.1% trifluoroacetic acid aqueous solution, mobile phase B solution: 0.1% trifluoroacetic acid + 99.9% acetonitrile solution (purchased from Acetonitrile Fisher Scientific). Flow rate: 1.0ml/min, sample volume 30μl, detection wavelength 220nm. Elution program: 0 to 5 minutes: 90% liquid A + 10% liquid B; 5 to 30 minutes: 90% liquid A/10% liquid B → 20% liquid A/80% liquid B.

2. Finally, freeze-dry the purified effective solution (lyophilizer Freezone Plus 6 model, LABCONCO manufacturer) to obtain the finished product.

3. Identification: Take a small amount of the finished peptides and perform HPLC to analyze their purity: high performance liquid chromatography (manufacturer SHIMADZU, Japan) and Venusi MRC-ODS C18 chromatographic column (4.6x150mm, Tianjin Bonna-Agela Technologies). Mobile phase A solution: 0.1% trifluoroacetic acid aqueous solution, mobile phase B solution: 99.9% acetonitrile + 0.1% trifluoroacetic acid solution, flow rate: 1.0ml/min, loading volume 10μl, detection wavelength 220nm. Elution program: 0~5min: 100% solution A; 5~30min: 100% solution A→20% solution A/80% solution B. It is required to measure purity greater than 95%. For specific methods, please refer to our authorized patent (Chinese patent ZL201410612382.3).

Identification of polypeptide molecular weight by MS method: Dissolve the polypeptide with qualified purity in water, add 5% acetic acid + 8% acetonitrile + 87% water to dissolve and test the molecular weight using electrospray ionization mass spectrometry. For the specific method, please refer to our authorized patent (Chinese patent ZL201410612382.3).

4. Seal the powdered peptides in sealed packages and store them away from light at -20°C.

3. Formation of dimers: Put a monomer peptide with a single cysteine inside the peptide chain at a concentration of 1 mg/ml in a disodium hydrogen phosphate aqueous solution with pH=9.5 and incubate it at 37°C overnight to form a homologous dimer. Polypeptide. For dimeric peptides with slightly poor solubility, centrifuge at 4000 rpm for 20 minutes to obtain the precipitate as pure dimeric peptide. Dissolve the precipitate in NaCl-PB solution (use disodium hydrogen phosphate to adjust the pH of physiological saline = 8.0) for use. . For the solubilized dimeric peptides or the above centrifugation supernatant, they were separated and identified by Sephadex G-25 chromatography (on a 2×60cm G-25 chromatography column and natural flow rate, using NaCl-PB solution as the flow term, dimers The component is the first peak, and the residual impurity component is the subsequent peak). Dimeric peptides can be identified by peptide PAGE electrophoresis or mass spectrometry without sulfhydryl reducing agent. For specific methods, please refer to our authorized patent (Chinese patent ZL201410612382.3).

4. The GLP-1R activator-like peptide monomer and its dimer were synthesized by our research laboratory or entrusted commercial companies. The inventor confirmed its structure through HPLC purity, ESI or laser flight mass spectrometry and cysteine oxidation. The GLP-1R activator monomer synthesized by the present invention is shown in Table 1, and the amino acid sequence of the homodimer peptide is shown in Table 2.

Table 1: Amino acid sequence of the GLP-1R activator-like peptide monomer synthesized by the present invention and its sustained hypoglycemic activity in a single injection of the same dose

Note: Tirzepatide in the table is a chimeric peptide of GIP-Exendin-4 peptide; Lixisenatide is also an allosteric version of Exendin 4; CFA (carbon fatty acid) or CFDA (carbon fatty diacid) is a carbon fatty acid or carbon fatty diacid; the above K[N-ε-(γ-Glu-N-α-CFA or CFDA)], K[N-ε-(2×AEEAC-γ-Glu-N-α-CFDA)] represents the lysine K side chain Fatty acyl or fatty diacid monoacyl glutamyl modification of the ε-amino group, the specific structure is shown in formula 1 or 2.

Example 2 Study on the persistence of the hypoglycemic effect of the GLP-1R activator of the present invention

1. Experimental method: Purchase normal KM mice from the Guangdong Provincial Animal Center for glucose tolerance test (OGTT): The glucose tolerance test of normal Kunming mice is used to screen the hypoglycemic activity and persistence of drugs. According to undifferentiated fasting blood glucose, male Kunming mice (5 weeks old) were divided into multiple groups (NaCl-PB group, Lixisenatide group, monomeric G9-G12 series and dimer 2G9-2G12 series groups) (n=6) . After two rounds of adaptation periods of 14 hours of food and 10 hours of fasting, KM mice were tested for glucose tolerance immediately after each 10-hour fast. After subcutaneous injection of drugs or monomers (dissolved with normal saline pH 6.5, and normal saline is used as the blank control) or dimeric peptides (dissolved with NaCl-PB solution pH 8.0, and NaCl-PB solution is generally used as the blank control) on the back. 30 min, the mice were gavaged with 5% glucose solution on time, and the tail blood glucose value of the mice was accurately measured after gavage. The blood glucose meter and blood glucose test strips are products of Bayer HeathCare LLC. The average blood sugar value of each group is used as the judgment standard: when the average blood sugar value of each group's OGTT is higher than the average blood sugar value of the blank control group on the same day for two consecutive times, the measurement is stopped. The duration of the blood sugar lower than the blank group during this period is the duration of the drug effect.

2. Experimental results

2.1 Oral glucose tolerance test

Thirty minutes after a single dose of glucose, blood glucose was measured from the tail blood of mice before (0 min) and 10, 20, 40, 60, and 120 min after a single oral administration of glucose. Normal saline was used in the blank control group (Figure 1). The results showed that at 10 minutes, compared with the Lixisenatide and G21 groups, the 2G33 group showed a significant increase in glucose (P<0.05). At 20 minutes, compared with the blank control group, the Lixisenatide and G21 groups showed significant decreases (P<0.05 or 0.01). Compared with the Lixisenatide and G21 groups, the 2G21 group showed a significant increase in glucose (P<0.05). At 40 minutes, compared with the blank control group, the Lixisenatide, G21 and G33 groups showed significant decreases (P<0.05 or 0.01). Compared with the blank control, Lixisenatide or G33 group, the 2G33 group showed a significant decrease or increase in glucose (P<0.05). At 60 minutes, compared with the blank control group, the G21 group showed a significant decrease (P<0.05); compared with the Lixisenatide and G21 groups, the 2G21 or 2G33 group showed a significant increase in glucose (P<0.05). The results showed that within the same period of time, the blood glucose value of the dimer was significantly higher than that of the monomeric peptide, indicating that the dimer significantly delayed absorption.

After a single dose, multiple OGTT tests (mOGTT) lasting for multiple days: 30 minutes after subcutaneous injection of drugs or monomeric or dimeric peptides on the back, mice were gavaged with 5% glucose solution on time, and the measurement was accurately performed 35 minutes after gavage. Rat tail blood glucose levels. Taking the average blood sugar as the judgment standard, the duration of hypoglycemic effect of Lixisenatide positive drug is 2 days, the duration of hypoglycemic effect of G9 series is 2-9 days, the G10 series is 2-11 days, the G11 series is 2-11 and the G12 series is 7-9 days. The dimer 2G9 series lasts for 4-21 days, the 2G10 series lasts for 4-22 days, the 2G11 series lasts for 6-21 days, and the 2G12 series lasts for 16-20 days. Each monomer group (shown in Table 1) lasts approximately 1/2 the duration of its corresponding dimer group (shown in Table 2). Comparison found that dimeric peptides with a disulfide bond formed at position 11 or 12 and a fatty acid modification of lysine epsilon-amino side chain at position 20 (among which 20-carbon fatty acids or fatty diacids are the best), have obvious As body weight decreases, the duration of hypoglycemia also increases significantly. Comparison found that the 11th peptide in the 2G9 series, the 20th and 21st in the 2G10 series, and the 33rd and 34th dimer peptides in the 2G11 series lasted up to 21-22 days. The 21st peptide (2G21) in the 2G10 series is the allosteric form of Exendin 4. It has high homology and the same side chain fatty acid modification as the sequence of the Lixisenatide positive control drug. Therefore, the 2G21 peptide was selected for the treatment and follow-up of type II diabetes (T2D) in vivo. experiment of. The mOGTT experiment needs to be carried out accurately, otherwise the results may be inconsistent.

Table 2 GLP-1R activator dimer sequence and duration of hypoglycemic activity of a single subcutaneous injection of the same dose

Note: CFA (carbon fatty acid) or CFDA (carbon fatty diacid) in the table is carbon fatty acid or carbon fatty diacid; the K[N-ε-(γ-Glu-N-α-CFA or CFDA)], K [N-ε-(2×AEEAC-γ-Glu-N-α-CFDA)] represents the fatty acyl or fatty diacid monoacyl glutamyl modification of the K side chain ε-amino group. The specific structure is as shown in Formula 1 or 2 Show. The dimer is connected by the disulfide bond formed between the cysteines in the monomer peptide, forming an H-type dimer; where "|" indicates the formation of a disulfide bond between two cysteine residues between the monomers. Sulfur bonds.

Example 3 Dimer therapeutic effect on type II diabetes model

1. Construction of type II diabetes (T2D) mouse model

C57Bl6/J mice were placed in an SPF-level environment with a standard diet and had free access to water. All experimental operations were performed in accordance with the guidelines for the ethics and use of laboratory animals. After being fed a standard diet for days, 5-week-old C57B16/J male mice were divided into 6 groups: NaCl-PB group, Placebo group (model control group), Lixisenatide group, and low, medium, and high-dose dimer peptide 2G21 group. The NaCl-PB group is the blank control and Placebo is the T2D model control, and they are injected with NaCl-PB solution. All T2D model groups were fed a 60 kcal% high-fat diet (D12492, Changzhou Shu Yi Shu Er Biotechnology Co., Ltd., Changzhou, China) until the end of the experiment, and the blank control group maintained a standard diet until the end of the experiment. Establishment method of diabetes model: After 4 weeks of high-fat feeding, mice were intraperitoneally injected with 75 mg/kg streptozotocin (STZ, Sigma Chemical Company, USA). 3 days later, mice were intraperitoneally injected with 50 mg/kg dose of STZ. 3 weeks later, Mice with blood glucose equal to or greater than 11mM were considered diabetic mice. These diabetic groups were followed by a 35-day treatment study on top of a high-fat diet.

2. Therapeutic effect on type II diabetes

Solubility of peptides: For Lys side chains containing fatty acid modified structures, monomeric peptides without Aib amino acids appear suspended in water, while their corresponding homodimer peptides are completely dissolved in water; for Lys side chains containing fatty acids Monomeric peptides with modified structures containing Aib amino acids or/and C-terminal amidation structures show complete solubility in water, while their corresponding homodimeric peptides are poorly soluble in water. Without Lys side chain fatty acid modification, both monomeric peptides and dimers are completely soluble in water. All dimeric peptides were dissolved in NaCl-PB (pH 8.0) for injection, and low, medium, and high doses of homodimer 2G21 were dissolved in NaCl-PB solution [Na ₂ HPO ₄ buffered saline solution (pH8.0)] for animal injection. Monomeric peptides are dissolved in physiological saline solution for injection (pH around 6.5).

Dosing concentration setting: Our preliminary experiments show that with a single subcutaneous injection of 0.624nmol/100μl Lixisenatide peptide, the time-effect relationship of multiple OGTTs over multiple days can be easily observed. Therefore, in all glucose tolerance experiments, normal Kunming mice were injected subcutaneously in the buttocks with a single dose of 0.624 nmol/100 μl Lixisenatide or monomeric peptide or dimeric peptide, and blood glucose was measured and weighed by tail-clipping at 9 o'clock every morning. For OGTT, the timing of drug administration, gastric administration, and blood glucose measurement for each animal needs to be accurate to the second. Lixisenatide was selected as the positive control and its administration method (subcutaneous administration) in T2D animal experiments.

Lixisenatide at 0.624nmol/100μl can induce the postprandial blood glucose level in the T2D diabetes model (postprandial blood glucose reaches 20mM) to reach 8-11mM. At this critical value, the effect-dose relationship between the positive drug Lixisenatide and GLP-1R dimer is easily observed. In the T2D treatment study, T2D model mice were injected subcutaneously into the buttocks with a dose of 100 μl each within 30 minutes. The blood glucose levels of the experimental mice were measured every five days. The entire test was completed within 40 minutes. The high, medium and low doses of the dimer 2G21 peptide are 1.873, 0.624 and 0.208nmol/100μL respectively. The dose of the positive drug Lixisenatide is 0.624nmol/100μl (the raw material drug is synthesized by a commercial company), which is injected once a day until the end of the 35-day experiment.

1. Body weight changes after T2D treatment: Before administration, there was no significant difference in the body weight of the T2D model group. After the experiment, compared with the NaCl-PB group, the Placebo group, Lixisenatide group, L-2G21 group (low dose), and M-2G21 group (medium dose) showed a significant increase in body weight (P<0.05 or 0.001), but H-2G21 There was no difference between the high dose group and the normal group, indicating that the treatment was effective. Compared with the Placebo group, the body weight of the Lixisenatide group and the H-2G21 group decreased significantly (P<0.05). The body weight of each 2G21 group decreased in a dose-dependent manner, and the M-2G21 group and the Lixisenatide group showed similar changes (Figure 2).

2. Hypoglycemic effect in T2D treatment: Compared with the NaCl-PB group, the Placebo, Lixisenatide, L- and M-2G21 groups had significantly higher fasting blood glucose values (Figure 3) (P<0.05 or 0.001), or Placebo, Lixisenatide, L- and M-, H-2G21 groups had significant high glycosylated hemoglobin (HbA _1c ) (Figure 4) (P<0.001), showing that the T2D model is successful. Compared with the Placebo group, the fasting blood glucose in the Lixisenatide and H-2G21 groups was significantly lower (P<0.05 or 0.01), or the HbA _1c in the Lixisenatide, M-2G21 and H-2G21 groups was significantly lower (P<0.05). Compared with the Lixisenatide group, fasting blood glucose in the L-2G21 group was significantly increased (P<0.05). After injecting the peptide, blood glucose levels decreased in a dose-dependent manner, and the effect became better with more times of administration. The blood glucose changes in the M-2G21 group were similar to those in the Lixisenatide group. HbA _1c and blood glucose values produce similar changes in T2D treatment.

3. Detection of blood biochemical indicators in T2D treatment: After the T2D treatment experiment, fasting insulin levels in the Placebo, Lixisenatide or L-2G21 group were significantly lower than those in the NaCl-PB group (P<0.01 or 0.001). Fasting insulin in each 2G21 group increased in a dose-dependent manner. Compared with the Lixisenatide group and the L-2G21 group, the insulin content in the M-2G21 group increased 2-3 times (P<0.05 or 0.01). Compared with the Placebo, Lixisenatide group or L-2G21 group, the insulin content in the H-2G21 group increased 2-4 times (P<0.05 or 0.001) (Figure 5), showing that equimolar concentrations of dimeric peptide induced 2-3 times insulin. secretion. The alanine aminotransferase (ALT) in the 2G21 group decreased in a dose-dependent manner, and the ALT levels in the H-2G21 group were lower than those in the NaCl-PB, Placebo, and L-2G21 groups (P<0.05) (Figure 6). The serum amylase in each 2G21 group decreased in a dose-dependent manner, but there was no statistical difference with the blank control or Placebo and Lixisenatide (P>0.05) (Figure 7).

From the above examples, we can draw the following conclusion: the homodimer series we developed can significantly increase the drug effect time. Studies have shown that dimer sequences show the most promising application prospects in rodent T2D models, such as the longest-lasting hypoglycemic effect and weight loss.

The structure-activity relationship shows that dimers without fatty acid modifications have the best solubility in water, and dimers with fatty acid-modified peptides containing Aib amino acid structures, even with C-terminal amidation structures, have slightly poorer solubility in water. These It is important data for drug preparation research, and is also the fundamental reason why GLP-1R activators with different structures in this study have different hypoglycemic activity durations—different spatial conformations, resulting in different physical and chemical properties, resulting in different hypoglycemic durations.

A single dose of the same dose was administered, and the results of a single OGTT experiment showed that the slow absorption of the dimer produced a longer hypoglycemic effect. The results of multiple OGTT experiments after a single dose of the same dose showed that the longer duration effect involves changes such as the second amino acid of the dimer, the disulfide bond position, Lys modified by symmetric fatty acids or fatty diacids, and C-terminal amidation. structure related. As shown in Table 2, the longest active dimer structures contain ² αAib, ¹¹ or ¹² Cys-Cys disulfide bonds, symmetric 20 fatty acyl or fatty diacid monoacyl-L-γ-glutamyl- ²⁰ lysine Or 20-carbon fatty acid diacyl monoacyl-γ-Glu-2×AEEAC- ²⁰ Lys and C-terminal amidation and other structures. The characteristics of these modifications are as follows: (1) αAib → ² Ala substitution produces longer activity; (2) Compared with other fatty acid modifications, Lys[2×AEEAC-γ-Glu-(N-α-20 carbon fatty diacid)] Modification achieves the best results; (3) C-terminal amidation significantly extends the activity; (4) The disulfide bond structure at position 12 or 11 in the dimer molecule shows the best activity. The activity of the monomeric peptide is only 1/2 that of the corresponding dimer.

In the T2D treatment experiment, the 2G21 group significantly reduced HbA _1c or fasting blood glucose (FPG) values in the diabetes model and had a significant hypoglycemic effect. The same molar concentration of 2G21 peptide and Lixisenatide had a significant effect on PPG (postprandial blood glucose) or FPG and HbA _1c. There is a similar reducing effect.

The body weight of the 2G21 group decreased in a dose-dependent manner, and the weight loss of the M-2G21 group and the Lixisenatide group were consistent. Better dimeric peptide structures for weight reduction involve structures such as ¹¹ or ¹² Cys-Cys disulfide bonds, symmetrical ²⁰ Lys [2×AEEAC-γ-Glu-20 carbon fatty acid] modification, and C-terminal amidation.

2G21 reduces alanine aminotransferase (ALT) in a dose-dependent manner, indicating that the drug has a strong protective effect on the liver.

In T2D treatment experiments, the insulin in the 2G21 group increased in a dose-dependent manner. Compared with Placebo, Lixisenatide and L-2G21, the M- or H-2G21 group induced 2-4 times higher insulin levels, so 2G21 has a better lowering effect. Sugar effect.

To sum up, the dimer peptide of the present invention can induce more insulin release, thereby producing better hypoglycemic effect.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (4)

1. A glucagon-like peptide-1 receptor (GLP-1R) activator-like peptide, characterized in that the specific sequence of said analog peptide is any one of the following:

①(HN ₂ )Y-αAib-EGTFTCDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

②(HN ₂ )Y-αAib-EGTFTCDYSI-αAib-LDK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]IAQKAFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

③(HN ₂ )Y-αAib-EGTFTCDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPSKKKKKK(NH ₂ ) Or;

④(HN ₂ )Y-αAib-EGTFTCDYSI-αAib-LDKIAQK[N-ε-(L-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

⑤(HN ₂ )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

⑥(HN ₂ )Y-αAib-EGTFTSDYCIALDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

⑦(HN ₂ )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(L-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

⑧(HN ₂ )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPSKKKKKK(NH ₂ ) Or;

⑨(HN ₂ )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(L-γ-Glu-N-α-18CFA)]AFVQWLIAGGPSSGAPPPS(NH ₂ ) Or;

⑩(HN ₂ )Y-αAib-EGTFTSDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPCSGAPPPS(NH ₂ ) Or;

(HN ₂ )Y-αAib-EGTFTSDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSCGAPPSKKKKKK(NH ₂ )。

2. a hypoglycemic peptide-like homodimer, characterized in that the dimer is formed by connecting identical monomers through disulfide bonds formed by cysteines, thus forming an H-type GLP-1R activator peptide-like homodimer, wherein the cysteines used for connection are positioned at the 8 th position or 11 th position or 32 rd position or 33 rd position on the dimer monomers; wherein the dimer of cysteine 11 is formed by the activator-like peptide monomer of claim 1, and the specific sequence is any one of the structures shown in the following:

wherein, the specific structure of the dimer of the 11 th cysteine is one of the following two types:

wherein, the specific structure of the dimer of the 32 nd cysteine is one of the following two types:

wherein, the specific structure of the dimer of the 33 rd cysteine is one of the following two types:

wherein \' represents the disulfide bond formed between the two cysteines.

3. Use of a GLP-1R activator like peptide according to claim 1, or a homodimer according to claim 2, for the preparation of a medicament for the treatment of hyperglycemia, diabetes and obesity.

4. A medicament for treating hyperglycemia, diabetes and obesity, characterized in that the medicament comprises the GLP-1R activator-like peptide or/and pharmaceutically acceptable salt thereof as an active ingredient; or the homodimer or/and pharmaceutically acceptable salt thereof as claimed in claim 2 as active ingredient.