CN116410298B

CN116410298B - Tri-agonism polypeptide compound and salt, pharmaceutical composition, medicament and application thereof

Info

Publication number: CN116410298B
Application number: CN202310659207.9A
Authority: CN
Inventors: 李忠碧; 姜薇
Original assignee: Norbotech Chengdu Biotechnology Co ltd
Current assignee: Norbotech Chengdu Biotechnology Co ltd
Priority date: 2023-06-06
Filing date: 2023-06-06
Publication date: 2023-08-11
Anticipated expiration: 2043-06-06
Also published as: CN116410298A

Abstract

The invention discloses a tri-excitation polypeptide compound and a salt, a medicinal composition, a medicament and application thereof, belonging to the technical field of polypeptide pharmaceutical chemistry. The amino acid sequence of the GLP-1, GIP and GCG tri-excitation polypeptide compound is as follows: BGS-3: haibHGTFTSDYSISIEbLEKIAQKAQQWLLEGGPSSGAPPPS-NH ₂ . The invention also discloses pharmaceutically acceptable salts, pharmaceutical compositions and medicaments of the GLP-1, GIP and GCG tri-agonism polypeptide compounds. The polypeptide compound has the weight-reducing effect at the same time, and can be used as an effective raw material for preparing weight-reducing medicines.

Description

Tri-agonism polypeptide compound and salt, pharmaceutical composition, medicament and application thereof

Technical Field

The invention relates to the technical field of polypeptide compounds, in particular to a tri-excitation polypeptide compound and salts, medicinal compositions, medicaments and uses thereof.

Background

The only therapeutic drugs currently available in China for patients with obesity or overweight (BMI is more than or equal to 24kg/m < 2 >) are orlistat capsules, which mainly reduce the weight by reducing the fat absorption in food, but have general weight reduction effects and can cause adverse drug reactions such as diarrhea, gastrointestinal discomfort, vitamin deficiency and the like, and the application of the therapeutic drugs in clinic is not wide. There is a far unmet clinical need for weight-reducing drugs in China.

GLP-1 receptor single-agonist weight-reducing drugs such as liraglutide and semraglutide are marketed abroad, wherein the weight of patients treated by the semraglutide is reduced by 12.4 percent in 68 weeks, and 69 percent of the weight of the patients is reduced by more than 10 percent, meanwhile, the global market of the GLP-1R/GIPR double-target drug telpopeptide is marketed in 2022 for more than 100 hundred million cents, the Gift first GLP-1R/GIPR double-target drug telpopeptide is marketed and achieves curative effect upgrading, the early data of the product CagriSema of the Norand Norde next generation is bright and pushed to the clinical stage 3, the clinical data of the first GLP-1R/GIPR/GCGR triple-agonist of the Gift is bright, but the domestic weight-reducing market needs more safe and effective weight-reducing drugs, and the GLP-1R/GIPR/GCGR triple-agonist of clinical research is still not clinically studied in China at present. The downstream signal transduction of GLP-1R, GIPR and GCGR receptor is complicated, positive and negative feedback regulation can exist on glycolipid metabolism, and the difficulty of designing an ideal multiple-activity hybrid peptide is increased. The field is to design a hybrid polypeptide with strong GLP-1R/GIPR/GCGR tri-agonist bioactivity, long half-life and low immunogenicity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a three-excited polypeptide compound, a salt, a medicinal composition, a medicament and application thereof, and a hybrid polypeptide with strong GLP-1R/GIPR/GCGR three-excited biological activity, long half-life and low immunogenicity is designed.

The aim of the invention is realized by the following technical scheme:

GLP-1, GIP and GCG tri-agonism polypeptide compound, the amino acid sequence of the polypeptide compound is:

BGS-3：

HAibHGTFTSDYSIAibLEKIAQKAFVQWLLEGGPSSGAPPPS-NH ₂ ；

wherein all Aib are 2-aminoisobutyric acid;

wherein the 20 th letter K of BGS-3 is a fatty acid linking position, wherein the side chain-modified K is K (AEEA-AEEA-gamma-Glu-CO- (CH) ₂ )n-COOH)；

Wherein n is a natural number, and n is more than or equal to 12 and less than or equal to 20.

Further, n is 14, 16, 18 or 20.

The invention also provides pharmaceutically acceptable salts of a class of GLP-1, GIP and GCG tri-agonistic polypeptide compounds. Pharmaceutically acceptable salts and common methods for preparing them are well known in the art.

Further, pharmaceutically acceptable salts of GLP-1, GIP and GCG tri-agonism polypeptide compounds are formed by the reaction of the polypeptide compound with any one of inorganic and organic acids.

Further, the salt is formed by GLP-1, GIP and GCG tri-agonist polypeptide compounds and one of the following compounds: hydrochloric acid, maleic acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectic acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, hydrobromic acid, salicylic acid, lauric acid, 2- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, lithonic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphate, aspartic acid, thiocyanic acid, or sulfosalicylic acid.

The invention also provides a pharmaceutical composition of GLP-1, GIP and GCG three-agonism polypeptide compounds, which takes the GLP-1, GIP and GCG three-agonism polypeptide compounds as effective raw materials, or takes pharmaceutically acceptable salts of the GLP-1, GIP and GCG three-agonism polypeptide compounds as effective raw materials, and also comprises a pharmaceutically acceptable carrier, diluent or excipient.

Further, the pharmaceutically acceptable carrier includes one or more of a binder, a lubricant, a disintegrant, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a flavoring agent, a buffer, a solubilizer, an isotonic agent.

The invention also provides a medicament prepared from the GLP-1, GIP and GCG tri-agonist polypeptide compound, wherein the medicament is any one of tablets, capsules, elixirs, syrups, lozenges, inhalants, sprays, injections, films, patches, powders, granules, blocks, emulsions, suppositories or compound preparations.

The invention also provides application of the GLP-1, GIP and GCG tri-agonist polypeptide compound, wherein the application is application of the GLP-1, GIP and GCG tri-agonist polypeptide compound serving as effective raw materials in preparing medicines for treating or preventing obesity, or in preparing medicines for treating or preventing fatty liver diseases, or in preparing medicines for treating or preventing nonalcoholic fatty liver diseases, or in preparing medicines for treating or preventing dyslipidemia, or in preparing medicines for treating or preventing metabolic syndrome.

The invention provides application of a pharmaceutically acceptable salt of the GLP-1, GIP and GCG tri-agonist polypeptide compound, wherein the application is application of the pharmaceutically acceptable salt of the polypeptide compound as an effective raw material in preparing medicines for treating or preventing obesity, or in preparing medicines for treating or preventing fatty liver diseases, or in preparing medicines for treating or preventing nonalcoholic fatty liver diseases, or in preparing medicines for treating or preventing dyslipidemia, or in preparing medicines for treating or preventing metabolic syndrome.

Throughout this specification, the following abbreviations are used in the following table:

the beneficial effects of the invention are as follows:

(1) The GLP-1, GIP and GCG tri-agonist polypeptide compounds of the present invention have higher agonistic activity at the GLP-1 receptor and GIP receptor than the natural ligand of each receptor, while having lower agonistic activity at the GCG receptor.

The GLP-1, GIP and GCG tri-excitation polypeptide compound provided by the invention is introduced with unnatural amino acid and fatty chain modification, has stable chemical property, is not easy to be degraded by DPP-IV and NEP in vivo, is not easy to be filtered by glomerulus, has obviously improved stability, and has the pharmacokinetic characteristic of supporting once-weekly administration.

(3) Compared with the existing GLP-1 receptor agonist, the GLP-1, GIP and GCG tri-agonism polypeptide compound has unexpected beneficial effects compared with the existing medicines in more effective promotion of weight reduction and weight gain prevention, reversion of insulin resistance and regulation of lipid metabolism.

Drawings

FIG. 1 is a graph showing the results of weight change in mice of different groups;

FIG. 2 is a graph showing the results of the weight change rate of mice of different groups;

FIG. 3 is a graph showing the body weight results of mice in each group at the end point;

FIG. 4 is a graph showing the results of daily feeding in mice of different groups;

FIG. 5 is a graph showing the cumulative food intake change results for different groups of mice;

FIG. 6 is a first graph of OGTT test results for different groups of mice;

FIG. 7 is a second graph of OGTT test results for different groups of mice;

FIG. 8 is a graph showing liver/fat weight ratio results for different groups of mice at endpoint;

FIG. 9 is a graph showing the biochemical detection results of blood of mice of different groups at the end point.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by a person skilled in the art without any inventive effort, are intended to be within the scope of the present invention, based on the embodiments of the present invention.

Example 1 (determination of agonistic Activity of GLP-1, GIP and GCG Tri-agonistic polypeptide Compounds on GLP-1 receptor, GCG receptor and GIP receptor)

The amino acid sequences of GLP-1, GIP and GCG tri-agonism polypeptide compound samples were:

BGS-3：

HAibHGTFTSDYSIAibLEKIAQKAFVQWLLEGGPSSGAPPPS-NH ₂ ；

wherein all Aib are 2-aminoisobutyric acid;

Wherein n is 16.

Agonism of the receptor by the polypeptide compounds is determined by a receptor function assay in which CHO cells stably express GLP-1 receptor, GCG receptor or GIP receptor and a Luciferase reporter gene comprising a cAMP response element, and the expression of the reporter gene Luciferase can be promoted by cAMP/PKA signaling pathway after stimulation of the stabilized cells by the polypeptide drug, and the activity of the stimulating drug is detected by detecting fluorescence produced by the Luciferase catalytic substrate.

Cells stably expressing the three receptors were split into T75 flasks and grown in medium (DMEM/10% fbs) overnight to near confluency, then the medium was removed, the cells were washed with PBS, digested for 3 min with 0.05% trypsin digest, then DMEM basal medium was added to terminate digestion, and cells were collected by centrifugation. Cell density was adjusted to 1X 10 with DMEM blank medium ⁵ Mu.l/ml, 100. Mu.l/well were inoculated into 96-well cell culture plates and incubated overnight at 37℃under 5% carbon dioxide.

And (3) drug treatment: pre-diluting the polypeptide sample to 5 mug/ml with a diluent to obtain a first dilution concentration, and sequentially diluting the polypeptide sample with the diluent in a 96-well plate for 8 times according to a 3-time ratio to obtain 9 concentrations; after dilution, each concentration of sample was added to the cell plate with 100. Mu.l of each well and double wells were made in parallel, and the dilution was used as a blank, and the mixture was incubated at 37℃in a 5% carbon dioxide incubator at about 5. 5 h.

Detection and result analysis: the luciferase detection kit is balanced to room temperature, 100 mu l of luciferase reagent in each hole is added into a cell plate, the reaction is carried out for 10 min at 200 rpm at room temperature under shaking and light shielding conditions, and the chemiluminescent RLU value is measured by a multifunctional enzyme-labeling instrument. EC50 was calculated using four-parameter logistic fit with chemiluminescent unit values (RLU) on the ordinate and sample concentrations on the abscissa using the microplate reader with its own software.

TABLE 1 determination of GLP-1, GCG and GIP receptor agonistic Activity of the Polypeptides

As shown in Table 1, BGS-3 has strong agonistic activity to all three receptors, especially GLP-1 receptor.

Example 2 (determination of serum stability with respect to GLP-1, GIP and GCG Tri-agonistic polypeptide Compounds)

BGS-3：

HAibHGTFTSDYSIAibLEKIAQKAFVQWLLEGGPSSGAPPPS-NH ₂ ；

wherein all Aib are 2-aminoisobutyric acid;

Wherein n is 18.

The test samples were incubated with human serum at 37℃for 0, 24, 48, 72 hours, and the serum stability of the polypeptide compounds was examined using the activity assay method of example 1.

TABLE 2 biological activity detection data for polypeptide serum stability

The relative percentage of this moment is EC at 0h from the corresponding receptor ₅₀ Data divided by the time corresponding to the receptor EC ₅₀ For example, the relative percentage of BGS-3-24h at the GLP-1 receptor is 151.0/104.5=144.5%.

As shown in Table 2, with the biological activity of the 0-hour sample of BGS-3 being 100%, the agonistic activity of BGS-3 at each time point on the three receptors was not affected, indicating that it was very stable in serum.

Example 3 (Effect of GLP-1, GIP and GCG Tri-agonism polypeptide Compounds on diet-induced obese (DIO) mouse body weight)

BGS-3：

HAibHGTFTSDYSIAibLEKIAQKAFVQWLLEGGPSSGAPPPS-NH ₂ ；

wherein all Aib are 2-aminoisobutyric acid;

Wherein n is 20.

Male C57BL/6J mice, weighing about 22g, were kept in 30 model groups and fed with high-fat feed for 18 weeks to prepare DIO mouse models. The blank 6 was fed with standard mouse feed (control standard diet) and was G1. Before the start of the administration, DIO mice in each group were randomly grouped according to body weight, and were divided into 4 groups of 6 groups each, G2, G3, G4, and G5, respectively.

After the test, the group G1 is fed by using a standard mouse feed, and the drug administration is PBS buffer solution; the group G2 is fed by using high-fat feed, and the drug administration is PBS buffer solution; group G3 was fed with a high fat diet and administered with a solution of feed 1 prepared from rope Ma Lutai; group G4 was fed with a high fat feed and the drug administered was a supply 2 solution made of telipopeptide; group G5 was fed with a high fat diet and administered with a solution of drug substance 3 prepared from the peptide compound of example 3.

Each group was dosed starting at D0 according to the experimental design. The administration date is: d0, D3, D7, D10, D14, D17, D21 and D24.

Dosing volume: mice were dosed with volume = 5 μl/g x mouse body weight (g) according to mouse body weight adjustment.

And (3) data acquisition:

(1) Mice were weighed daily during the drug effect period;

(2) Detecting the food intake of each group of mice every day, and calculating average daily food intake and accumulated food intake;

(3) 24h after the first dose, OGTT test (glucose dose: 2 g/kg,0, 15, 30, 60, 120 min) was performed;

(4) The last administration was followed by a body fat ratio test.

Sample collection: d28 test endpoint, test endpoint treatment was as follows:

(1) Weighing liver tissue, quickly freezing with liquid nitrogen, temporarily storing at-80 ℃, calculating the weight-to-weight ratio of the liver, and processing the liver sample after confirming the data by a consignor without storing;

(2) Mouse adipose tissue was collected: (1) the inguinal fat, (2) epididymal fat, (3) perirenal fat, (4) mesenteric fat, (5) subscapular brown fat, and the five parts were respectively weighed and recorded, the fat weight ratio of each part was calculated, and the white fat ((1) - (4) added) weight ratio was calculated.

Referring to fig. 1-9, the results of the study showed that: compared with the model control group G2 (PBS), the group G5 (BGS-3) can obviously reduce the weight of mice and OGTT after administration, obviously reduce the subcutaneous fat weight ratio of inguinal, the mesenteric fat weight ratio and the brown fat weight ratio of scapula, and obviously reduce the levels of CHOL and LDL-C of plasma. Each BGS-3 group has inhibiting effect on ingestion of mice and improving effect on ALT and AST.

The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

Claims

GLP-1, GIP and GCG tri-agonism polypeptide compound, characterized in that the polypeptide compound is named BGS-3, and the amino acid sequence of the polypeptide compound is:

HAibHGTFTSDYSIAibLEKIAQKAFVQWLLEGGPSSGAPPPS-NH ₂ ；

wherein all Aib are 2-aminoisobutyric acid;

wherein the 20 th letter K of BGS-3 is a fatty acid linking positionWhere the side chain modified K is K (AEEA-AEEA-gamma-Glu-CO- (CH) ₂ )n-COOH)；

Wherein n is a natural number, and n is more than or equal to 12 and less than or equal to 20.
2. The GLP-1, GIP and GCG tri-agonism polypeptide compound of claim 1, characterized in that: the n is 14, 16, 18 or 20.
3. A pharmaceutically acceptable salt of a GLP-1, GIP and GCG tri-agonist polypeptide compound of claim 1.
4. The pharmaceutically acceptable salt of a GLP-1, GIP and GCG tri-agonist polypeptide compound of claim 3, wherein: formed by the reaction of a GLP-1, GIP and GCG tri-agonistic polypeptide compound according to claim 1 or 2 with any one of inorganic and organic acids.
5. The pharmaceutically acceptable salt of the GLP-1, GIP and GCG tri-agonist polypeptide compound of claim 4, wherein: the salt is formed by GLP-1, GIP and GCG tri-agonist polypeptide compounds and one of the following compounds: hydrochloric acid, maleic acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectic acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, hydrobromic acid, salicylic acid, lauric acid, 2- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, lithonic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphate, aspartic acid, thiocyanic acid, or sulfosalicylic acid.
A pharmaceutical composition of GLP-1, GIP and GCG tri-agonist polypeptide compounds, characterized in that the pharmaceutical composition takes GLP-1, GIP and GCG tri-agonist polypeptide compounds according to claim 1 or 2 as effective raw materials, or takes pharmaceutically acceptable salts of GLP-1, GIP and GCG tri-agonist polypeptide compounds according to any one of claims 3-5 as effective raw materials, and further comprises pharmaceutically acceptable carriers, diluents or excipients.
7. The pharmaceutical composition of GLP-1, GIP and GCG tri-agonist polypeptide compound of claim 6, characterized in that: the pharmaceutically acceptable carrier comprises one or more of a binder, a lubricant, a disintegrant, a dispersing agent, a stabilizer, a suspending agent, a colorant, a flavoring agent, a buffering agent, a solubilizer, and an isotonic agent.
8. The pharmaceutical formulation of GLP-1, GIP and GCG tri-agonism polypeptide compound of any one of claims 1 or 2, wherein the pharmaceutical formulation is a tablet, capsule, elixir, syrup, lozenge, inhalant, spray, injection, film, patch, powder, granule, block, emulsion, suppository or compound formulation.
9. Use of a GLP-1, GIP and GCG tri-agonist polypeptide compound according to any one of claims 1 or 2, characterized in that it is a use of GLP-1, GIP and GCG tri-agonist polypeptide compound as an effective raw material for the preparation of a medicament for the treatment of obesity, or for the preparation of a medicament for the treatment of fatty liver disease, or for the preparation of a medicament for the treatment of dyslipidemia, or for the preparation of a medicament for the treatment of metabolic syndrome.
10. Use of a pharmaceutically acceptable salt of a GLP-1, GIP and GCG tri-agonist polypeptide compound according to any one of claims 3 to 5, characterized in that the use is the use of a pharmaceutically acceptable salt of a GLP-1, GIP and GCG tri-agonist polypeptide compound as an effective raw material for the manufacture of a medicament for the treatment of obesity, or for the manufacture of a medicament for the treatment of fatty liver disease, or for the manufacture of a medicament for the treatment of dyslipidemia, or for the manufacture of a medicament for the treatment of metabolic syndrome.