WO2019037393A1 - Polypeptide compound having high affinity for glp-1 receptor, preparation method therefor and use thereof - Google Patents

Abstract

The present invention provides a polypeptide compound having high affinity for GLP-1 receptors, the molecular structure thereof being as shown in the following formula (I), wherein R represents Evans blue or a derivative group thereof. The polypeptide compound of the present invention has the advantages of stable structure, simple preparation, significant therapeutic effect, long-term drug efficacy, convenient storage, etc. The present invention further provides a preparation method for the polypeptide compound and use thereof in preparing a medicament for diagnosing, preventing or treating GLP-1/GLP-1 receptor pathway-related diseases.

Description

Polypeptide compound having high affinity to GLP-1 receptor, preparation method and application thereof Technical field

The invention belongs to the field of biomedicine and pharmaceutical preparations, and particularly relates to a polypeptide compound having high affinity to a GLP-1 receptor, a preparation method thereof and a disease thereof related to the preparation of a GLP-1 or GLP-1 receptor pathway. Use in diagnostic, prophylactic or therapeutic drugs.

Background technique

The main function of glucagon peptide-1 (GLP-1) is to stimulate insulin secretion, inhibit glucagon secretion, promote satiety, inhibit gastrointestinal motility, increase glucose intake and reduce body weight. In non-insulin-dependent diabetes mellitus (NIDDM), GLP-1 promotes the growth of new cells and restores insulin secretion without lowering blood sugar. Furthermore, GLP-1 does not cause any toxic side effects by the route of administration by injection. Therefore, GLP-1 plays an important role in the treatment of type 2 diabetes. In addition, GLP-1 receptor is expressed in both cardiomyocytes and endothelial cells. GLP-1 can effectively protect myocardial function by activating GAMP-1 receptor cAMP, phosphatidylinositol 3-kinase and activating antioxidant enzymes. And fibrosis. Therefore, GLP-1 has potential therapeutic effects on myocardial ischemia, myocardial ischemia-reperfusion injury, chronic heart failure and secondary myocardial infarction. The treatment of myocardial disease with GLP-1 as a lead compound has also become a new drug development. hot spot.

However, GLP-1 is a substrate for endogenous dipeptidyl-IV (DPP-IV), which degrades the N-terminal histidine (7)-alanine of GLP-1 (8). The peptide bond inactivates GLP-1, resulting in a GLP-1 half-life in vivo of only 1-2 minutes. In order to reduce the degradation of GLP-1 and prolong the retention time of GLP-1 in plasma, it is necessary to develop a ligand that uses a DPP-IV inhibitor, or a reaction using a GLP-1 receptor and a GLP-1 derivative, such as Exendin-4. . Exendin-4 has 39 amino acids and has high homology with GLP-1. It can effectively inhibit DPP-IV hydrolysis and extend the half-life in vivo to 2-4 hours. For example, the commercial drug Exenatide can effectively control blood sugar levels, but the twice-daily administration brings a lot of inconvenience to the patient. In order to further improve the blood half-life of the drug and prolong the efficacy, the development of the derivative of Exendin-4 is mainly concentrated in two directions: First, the PEG with a molecular weight of 5K-12K Dalton is modified in the lysine or half of Exendin-4. On cystine. Although this extends the half-life, it reduces the binding of the drug molecule to the receptor and reduces the biological activity of the drug. Second, the drug molecule is combined with albumin with a long half-life to achieve long-term pharmacodynamic effects of the drug. For example, the sixteen-carbon chain modified drug molecule Liraglutide can bind to albumin to achieve one-time injection; the exendin-4 covalently modified albumin molecular drug molecule Albiglutide has a half-life of 4-7 days, but Albiglutide is a protein product that is not conducive to storage and is too expensive. These also limit clinical applications.

Evans Blue (EB), also known as Evans Blue, binds to albumin in vitro and in vivo to form EB-albumin complexes, effectively increasing the in vivo half-life of EB molecules. Through chemical modification, the amino-functionalized EB molecule (EB-NH ₂ ) is linked to different reaction sites, and the molecules with different functions are covalently modified, for example, the drug molecule, NOTA, DOTA, etc. are modified on the EB, PET imaging and treatment for tumors and lymph nodes.

Based on the above considerations, the prior art proposes a method for preparing a GLP-1 receptor affinity polypeptide by modifying Evans Blue or a derivative thereof on Exendin-4, and preliminary studies have shown that The polypeptide retains the natural biological activity of Exendin-4 and also prolongs the half-life in vivo, reducing the dose and number of administrations in the treatment of type 2 diabetes. However, the study found that the stability of such peptides needs to be improved, which can not meet the needs of long-term storage and transportation of drugs, leading to limited clinical application.

Summary of the invention

The primary object of the present invention is to provide a polypeptide having high affinity for the GLP-1 receptor, which has a good DPP-IV inhibitory effect, high stability, and can maintain a stable molecular structure for a long time, and is effective. Increase the half-life in the body.

The above object of the present invention is achieved by the following technical solutions:

Provided is a polypeptide compound having high affinity for the GLP-1 receptor, the molecular structure of which is represented by the following formula (I):

Wherein R represents an Evans blue or a derivative thereof.

In the embodiment of the present invention, the Evans blue or a derivative thereof may be derived from Evans blue or a derivative thereof modified with different chain lengths. In a preferred embodiment, the Evans blue or a derivative thereof is selected from the group consisting of Evans Blue Maleimide, Evans Blue Aldehyde, Evans Blue Pyridyl Dithiol or Evans Blue Iodoacetyl One.

In a most preferred embodiment of the present invention, the R of the formula (I) is an Evans blue maleimide group, that is, the molecular structure of the polypeptide compound having high affinity for the GLP-1 receptor is as follows Formula (II):

Another object of the present invention is to provide a method of preparing a polypeptide compound having high affinity for the GLP-1 receptor of the present invention.

The preparation method comprises: using Evans Blue or a derivative thereof and [Leu] ¹⁴ Exendin-4 as a raw material, the Evans blue or a derivative thereof having a group reactive with a thiol group, The [Leu] ¹⁴ Exendin-4 terminal is modified with a cysteine Cys-, and Evans blue or a derivative thereof is modified at the [Leu] ¹⁴ Exendin-4 terminal cyste by a coupling reaction between the starting materials. On the sulfhydryl group of the amino acid Cys, a polypeptide compound having high affinity for the GLP-1 receptor according to the present invention is obtained.

The preferred method of the present invention specifically includes the following steps:

(1) Evans blue or a derivative thereof having a cysteine Cys-[Leu] ¹⁴ Exendin-4 and a group reactive with a thiol group in a sodium acetate solution in the presence of a reducing agent The coupling reaction occurs in the dark, and the reaction is carried out at room temperature for 1-3 hours to obtain a reaction mixture;

(2) lyophilizing the reaction mixture of the step (1) to obtain a lyophilized mixture;

(3) separating and purifying unreacted Evans blue or a derivative thereof, unreacted [Leu] ¹⁴ Exendin-4, and a coupling product from the lyophilized mixture obtained in the step (2), the coupling product That is, the polypeptide compound having high affinity to the GLP-1 receptor of the present invention.

In the method of the present invention, the Evans blue or a derivative thereof having a group reactive with a thiol group as described in the step (1) may be Evans blue or a derivative thereof having a different chain length modification, and a group that reacts with a thiol group.

In a further preferred method of the present invention, the group reactive with a thiol group may be selected from any of a maleimide group, a pyridyldithiol group, an iodoacetyl group or an aldehyde group. One.

In a further preferred preparation method of the present invention, the concentration of the sodium acetate solution in the step (1) is 50 mM; the reducing agent is NaCNBH ₃ , and the reducing agent is added in a ratio of 20 mM.

In a further preferred preparation method of the present invention, the end of the step (1) is modified with a cysteine Cys-[Leu] ¹⁴ Exendin-4 and an Evans blue having a group reactive with a thiol group or a derivative thereof. The molar ratio of the reaction was 1:1.

In a further preferred preparation method of the present invention, the coupling reaction time described in the step (1) is 2 hours.

In the method of the present invention, the separation of unreacted material from the lyophilized mixture described in step (3) can be removed by a typical dialysis method, for example, by dissolving the lyophilized mixture in a suitable buffer for dialysis. There is no particular limitation on the buffer. Usually used in the field, PBS (phosphate buffer) can be used, the selected buffer should match the reaction conditions of Evans blue, depending on the functional group of Evans blue (maleic anhydride requires pH6) .5-7.5; pyridyldithiol or iodoacetyl requires pH alkaline, 7.0-8.5). The reaction system can be isolated and purified using size exclusion chromatography or reversed-phase high performance liquid chromatography.

Still another object of the present invention is to provide a use of the polypeptide compound having high affinity for the GLP-1 receptor for the preparation of a medicament for diagnosing, preventing or treating a disease associated with the GLP-1/GLP-1 receptor pathway.

The diagnosis includes imaging diagnosis and efficacy monitoring of diseases associated with the GLP-1/GLP-1 receptor pathway.

In a preferred embodiment of the invention, the GLP-1/GLP-1 receptor pathway associated disease is type II diabetes or myocardial infarction. Wherein, the polypeptide compound achieves prevention and treatment of type 2 diabetes by promoting excessive secretion of insulin; and activates cAMP and phosphatidylinositol 3-kinase by GLP-1 receptor to treat myocardial ischemic injury; The polypeptide compound is also capable of activating an antioxidant enzyme to reduce fibrosis caused by myocardial ischemia and promote functional recovery of the left ventricle.

In a preferred embodiment of the present invention, the polypeptide compound of the present invention is prepared as an active ingredient as an oral preparation or an injection.

The oral preparation can be either solid or liquid. The solid oral preparation dosage form may be a tablet, a pill, a powder, a granule or a capsule, etc., and usually requires at least one excipient ingredient such as starch, calcium carbonate, sucrose, lactose or gelatin; in addition to the auxiliary ingredients, there is lubrication. For example, use magnesium stearate or talc. The oral preparation in liquid form may be a suspension, a liquid for use in the body, an emulsion or a syrup; these oral dosage forms may be supplemented with a wetting agent, a sweetener, a flavoring agent, a preservative, or a simple diluent such as a liquid or liquid paraffin. .

The injection may be a sterile aqueous solution, a nonaqueous solvent, a suspension, an emulsion, a lyophilizate or a suppository. Non-aqueous solvents or suspending agents may contain propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and/or injectable esters such as ethyl oleate.

The amount of the preparation prepared in the present invention depends on various factors including sex, age, body weight, physical condition, diet, time and route of administration, metabolic rate, and severity of the disease. In general, the effective dose of the drug to be completely absorbed within one to two weeks is observed by different routes of administration. The formulations of the invention may be administered once, or multiple times a day, within the daily effective dosage range. Compared with the prior art, the polypeptide compound of the present invention retains the natural biological activity of Exendin-4, and has a longer in vivo half-life than the unmodified Exendin-4, and has good pharmacokinetic and pharmacological effects. It is worth mentioning that the amino acid sequence derived from [Leu] ¹⁴ Exendin-4 in the present invention brings good structural stability to the polypeptide compound, and is obtained in terms of molecular structure stability compared with other existing similar polypeptides. Significant improvement.

In the preparation method of the present invention, Evans Blue or a derivative thereof is modified by a coupling reaction to a thiol group of C cysteine Cys at the end of [Leu] ¹⁴ Exendin-4, and [Leu] ^{14 is} achieved. The immobilized Evans blue modification of Exendin-4 polypeptide is simple and easy to prepare.

In conclusion, the polypeptide of the invention has the advantages of simple preparation, obvious curative effect, long-lasting effect, stability, and convenient storage.

DRAWINGS

Figure 1 is a graph showing the purity of maleic dimethylenediamine prepared in the step a of Example 1;

Figure 2 illustrates the molecular weight of maleic dimethylenediamine prepared in step a of Example 1;

Figure 3 is a purity diagram of the maleimide Evans blue prepared in the step b of Example 1.

Figure 4 illustrates the molecular weight of the maleimide Evans blue prepared in step b of Example 1.

Figure 5 is a purity diagram of the polypeptide compound prepared in the step 2) of Example 1;

Figure 6 is a graph showing the molecular weight of the polypeptide compound prepared in the step 2) of Example 1;

Figure 7 is a graph showing the results of the stability determination of the polypeptide compound prepared in the step 2) of Example 1 and the comparative polypeptide;

Figure 8 is a graph showing the results of pharmacodynamic analysis of a mouse model of the polypeptide compound prepared in the step 2) of Example 1.

Figure 9 is a graph showing the results of pharmacodynamic analysis of the cynomolgus monkey model of the polypeptide compound prepared in the step 2) of Example 1.

Detailed ways

The present invention is specifically described by the following examples, which are only used to further illustrate the present invention, and are not to be construed as limiting the scope of the present invention. Improvements and adjustments are within the scope of the invention.

[Leu] ¹⁴ Exendin-4 used in the present invention can be obtained by chemical synthesis or genetic engineering recombinant expression of the prior art.

Example 1

1) Preparation of maleimide Evans blue derivatives:

a. Preparation of the intermediate maleic acid dimethylaniline Mal-tolidine

3-Maleimidopropionic acid (10.0 mmol), HATU (20 mmol) and DIPEA (50.0 mmol) were sequentially added to a solution of 3,3'-dimethylbenzidine tolidine (50.0 mmol) in acetonitrile (500 mL) . After stirring at room temperature for 48 h, the reaction mixture was filtered and evaporated mjjjd The residue was purified on a silica gel column to afford a pale-yellow intermediate, </ RTI> <RTIgt; The reaction process is as follows:

b. Preparation of Evans Blue (MEB) with maleimide groups

The maleic dimethylenedeidine Mal-tolidine (5.0 mmol) obtained in the step a was dissolved in a mixed solvent of acetonitrile and water to obtain a yellow solution. HCl was slowly added dropwise to the above Mal-tolidine solution under ice bath conditions. After stirring for 10 min, a solution of NaNO ₂ (20.0 mmol) was slowly added dropwise and stirring was continued for 30 min to obtain a yellow diazonium salt solution. NaHCO ₃ (30.0 mmol) and 1-amino-8-naphthol-2,4-disulfonic acid monosodium hydrate (5.0 mmol) were each dissolved in ice water. Then, the freshly prepared diazonium salt solution was slowly added dropwise to the above solution, and stirring was continued for 2 hours in an ice bath to obtain a purple solution. The purple solution was dialyzed against pure water and directly freeze-dried to obtain a purple powder, Mal-C3-EB (MEB), which is shown in Figures 3 and 4, and the yield was about 80%. The reaction process is as follows:

2) Preparation of Evans blue modified [Leu] ¹⁴ Exendin-4

Step 1) Prepare a solution of 5 mL of [Leu] ¹⁴ Exendin-4 (1 mmol) dissolved in 50 mM sodium acetate solution (50 mM, pH 5.5) in MEB (1 mmol) sodium acetate solution (50 mM, pH 5.5), and then A 20 mM NaCNBH ₃ reducing agent was added. The reaction was carried out at room temperature for 2 hours in the dark. The reaction was stopped with 0.1% aqueous trifluoroacetic acid (TFA) to obtain a reaction mixture, and the reaction mixture was lyophilized to obtain a lyophilized mixture, and then the lyophilized mixture was dissolved in PBS buffer at pH 6.5, and removed by dialysis. The reaction material finally gave [Eu] ¹⁴ Exendin-4 modified by Evans Blue. The purified Evans blue modified [Leu] ¹⁴ Exendin-4 was characterized as shown in Figures 5 and 6, with a retention time of 22.5 minutes.

The sequence of [Leu] ¹⁴ Exendin-4 used in this step is:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Leu-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys-NH ₂ .

The reaction process of this step is as follows:

Stability Analysis of [Met] ¹⁴ Exendin-4 Modified by Evans Blue

To test the stability of the polypeptide compound prepared in Example 1, we selected [Met] ¹⁴ Exendin-4, also modified by MEB, as a comparative polypeptide in which the modified [Met] ¹⁴ Exendin-4 sequence was:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys-NH ₂ .

The solutions of the two compounds were each placed at 37 ° C for 36 hours. The stability of the two solutions was monitored by liquid phase and mass spectrometry. As shown in Figure 7, after 36 hours of placement, the molecular weight of MEB-modified [Met] ¹⁴ Exendin-4 (comparative peptide) showed a new peak [m/z = 4999], which is attributed to the methionine on the peptide methionine. Oxidation product sulfoxide. On the other hand, there was no significant change in the molecular weight of the MEB-modified [Leu] ¹⁴ Exendin-4 of Example 1. This indicates that the Leu substitution of Met effectively increases the molecular stability of the polypeptide compound.

Study on the efficacy of small animal experiments

The efficacy of several drugs in type 2 diabetes was compared using a small animal model. In the type II diabetes model mice, the natural Exendin-4, the commercial Albiglutide, and the polypeptide compound prepared in Example 1 were separately injected into the mice by subcutaneous injection, and blood was taken at regular intervals, and the blood glucose concentration was monitored and quantified by an ELISA kit. Figure 8 shows. The results show that the polypeptide compound of Example 1 of the present invention is capable of maintaining blood glucose at a lower level than natural Exendin-4 and commercial Albiglutide for a longer period of time.

Study on the efficacy of large animal experiments

Using a large animal model to compare the efficacy of several drugs in type 2 diabetes, using the type II diabetic cynomolgus monkey as an experimental model, the commercial Albiglutide and the polypeptide compound prepared in Example 1 were injected subcutaneously into the type II diabetes model crab. In the monkey body, blood was taken periodically, and the blood glucose concentration was monitored and quantified by an ELISA kit. The result is shown in Figure 9. The results showed that the polypeptide compound prepared in Example 1 was superior to Albiglutide in the blood sugar control ability of the type II diabetes model cynomolgus monkey.

Example 2

A solution prepared by dissolving 5 mL of [Leu] ¹⁴ Exendin-4 (1 mmol) in 50 mM sodium acetate solution (50 mM, pH 5.5) was added to an aldehyde-containing Evans blue (1 mmol) sodium acetate solution (50 mM, pH 5.5). Then, 20 mM NaCNBH ₃ reducing agent was added. The reaction was carried out for 2 hours at room temperature in the dark. The reaction was stopped with 0.1% aqueous trifluoroacetic acid (TFA), the reaction mixture was lyophilized to obtain a lyophilized mixture, and then the lyophilized mixture was dissolved in PBS buffer at pH 7.0, and unreacted by dialysis. The substance, which finally got [Eu] ¹⁴ Exendin-4 modified by Evans Blue.

Example 3

Add 5 mL of [Leu] ¹⁴ Exendin-4 (1 mmol) in 50 mM sodium acetate solution (50 mM, pH) to Evans blue (1 mmol) sodium acetate solution (50 mM, pH 5.5) with pyridine dithiol group. 5.5) The resulting solution was then added with 20 mM NaCNBH ₃ reducing agent. The reaction was carried out for 2 hours at room temperature in the dark. The reaction was stopped with 0.1% aqueous trifluoroacetic acid (TFA), the reaction mixture was lyophilized to obtain a lyophilized mixture, and then the lyophilized mixture was dissolved in PBS buffer at pH 8.5, and unreacted by dialysis. The substance, which finally got [Eu] ¹⁴ Exendin-4 modified by Evans Blue.

Example 4

5 mL of [Leu] ¹⁴ Exendin-4 (1 mmol) in Evans Blue (1 mmol) sodium acetate solution (50 mM, pH 5.5) with iodoacetyl group dissolved in 50 mM sodium acetate solution (50 mM, pH 5.5) The resulting solution was then added with 20 mM NaCNBH ₃ reducing agent. The reaction was carried out for 2 hours at room temperature in the dark. The reaction was stopped with 0.1% aqueous trifluoroacetic acid (TFA), the reaction mixture was lyophilized to obtain a lyophilized mixture, and then the lyophilized mixture was dissolved in PBS buffer at pH 7.5, and unreacted by dialysis. The substance, which finally got [Eu] ¹⁴ Exendin-4 modified by Evans Blue.

Claims (10)

1. A polypeptide compound having high affinity for the GLP-1 receptor, the molecular structure of which is represented by the following formula (I):

   

   Wherein R represents an Evans blue or a derivative thereof.
2. The polypeptide compound according to claim 1, wherein the Evans blue or a derivative thereof is selected from the group consisting of Evans Blue Maleimide, Evans Blue Aldehyde, Evans Blue Pyridyl Dithiol or Evans Any of the blue iodoacetyl groups.
3. The polypeptide compound according to claim 1, wherein R of the formula (I) is an Evans blue maleimide group, that is, the polypeptide having high affinity for the GLP-1 receptor. The molecular structure of the compound is shown in the following formula (II):

   
4. A method for producing a polypeptide compound having high affinity for a GLP-1 receptor according to claim 1, characterized in that Evans Blue or a derivative thereof and [Leu] ¹⁴ Exendin-4 are used as a raw material The Evans blue or a derivative thereof has a group reactive with a thiol group, and the [Leu] ¹⁴ Exendin-4 terminal is modified with a cysteine Cys- by coupling between the raw materials In response, Evans blue or a derivative thereof is modified on the thiol group of [Leu] ¹⁴ Exendin-4 terminal cysteine Cys to obtain the above-described polypeptide compound having high affinity to the GLP-1 receptor.
5. The method of claim 4, comprising the steps of:

   (1) Evans blue or a derivative thereof having a cysteine Cys-[Leu] ¹⁴ Exendin-4 and a group reactive with a thiol group in a sodium acetate solution in the presence of a reducing agent The coupling reaction occurs in the dark, and the reaction is carried out at room temperature for 1-3 hours to obtain a reaction mixture;

   (2) lyophilizing the reaction mixture of the step (1) to obtain a lyophilized mixture;

   (3) separating and purifying unreacted Evans blue or a derivative thereof, unreacted [Leu] ¹⁴ Exendin-4, and a coupling product from the lyophilized mixture obtained in the step (2), the coupling product That is, the polypeptide compound having high affinity to the GLP-1 receptor.
6. The method according to claim 4 or 5, wherein the group reactive with the thiol group is selected from the group consisting of a maleimide group, a pyridyl dithiol group, an iodoacetyl group or an aldehyde. Any of the bases.
7. The method according to claim 5, wherein the end of the step (1) is modified with a cysteine Cys-[Leu] ¹⁴ Exendin-4 and an Evans blue having a group reactive with a thiol group. The reaction molar ratio of the derivative or the derivative thereof is 1:1; the sodium acetate solution concentration in the step (1) is 50 mM; the reducing agent in the step (1) is NaCNBH ₃ , and the addition ratio is 20 mM; the step (1) The coupling reaction time was 2 hours.
8. Use of the polypeptide compound having high affinity for the GLP-1 receptor according to claim 1 for the preparation of a medicament for diagnosing, preventing or treating a disease associated with the GLP-1/GLP-1 receptor pathway.
9. The use according to claim 8, characterized in that the GLP-1/GLP-1 receptor pathway-associated disease is type II diabetes or myocardial infarction.
10. The use according to claim 8, wherein the polypeptide compound according to claim 1 is prepared as an active ingredient as an oral preparation or an injection.

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