CN116589536B - Long-acting GLP-1/GIP receptor dual agonist and application thereof - Google Patents
Long-acting GLP-1/GIP receptor dual agonist and application thereof Download PDFInfo
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- CN116589536B CN116589536B CN202310562947.0A CN202310562947A CN116589536B CN 116589536 B CN116589536 B CN 116589536B CN 202310562947 A CN202310562947 A CN 202310562947A CN 116589536 B CN116589536 B CN 116589536B
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- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
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Abstract
A long-acting GLP-1/GIP receptor dual agonist and application thereof, wherein the N-terminal amino acid His of the receptor dual agonist sequence is consistent with natural GLP-1, and the commonly used Tyr is not adopted, so that better GLP-1 receptor agonistic activity and hypoglycemic activity are brought. In addition, the sequence structure of the dual agonist of the invention ensures that the GLP-1/GIP receptor dual agonist has strong GLP-1 receptor agonism and weaker GIP receptor agonism, and unexpectedly leads the GLP-1/GIP receptor dual agonist of the invention to have more excellent hypoglycemic, weight-reducing and lipid-regulating effects compared with the GLP-1/GIP receptor dual agonist on the market, thereby bringing about unexpected beneficial effects; the preparation method has great potential in preparing medicaments for treating metabolic syndrome, such as diabetes, obesity, nonalcoholic fatty liver disease, nonalcoholic fatty hepatitis, dyslipidemia and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a long-acting GLP-1/GIP receptor dual agonist and application thereof.
Background
According to the latest version of the international diabetes union (IDF) data, the number of people suffering from diabetes in China (20-79 years old) reaches 1.4 hundred million, and is the first place worldwide. Diabetes is largely classified into type 1 diabetes (T1 DM) and type 2 diabetes (T2 DM), with T2DM accounting for over 90% of adult diabetics. Studies show that overweight and obesity can obviously increase the incidence risk of T2DM, and for patients with T2DM accompanied by obesity, the blood sugar is reduced, the weight is reduced, the disease course development of T2DM can be controlled more effectively, and the complication risk and disability/death rate can be obviously reduced. The most effective current method for treating obesity and T2DM is still bariatric surgery (e.g., roux-en-Y gastric bypass surgery), but patients with such treatments suffer from a significant surgical risk. Therefore, there is still a great need for therapeutic drugs that can effectively treat T2DM with good weight loss.
Glucagon-like peptide-1 (GLP-1) is an incretin secreted by intestinal L cells in humans in response to nutrient intake, which effects its various biological effects by agonizing the GLP-1 receptor. The most important physiological function of GLP-1 is to promote insulin secretion in a glucose-dependent manner to lower blood glucose, so GLP-1 does not cause hypoglycemia in patients during use. In addition to the hypoglycemic effect, GLP-1 can stimulate differentiation and proliferation of pancreatic beta cells, prevent beta cell apoptosis and improve the process of T2DM from the source. In addition, GLP-1 can also increase satiety and suppress appetite, delay gastrointestinal motility and gastric emptying by acting on hypothalamic feeding centers, and has weight-reducing effect. However, native GLP-1 is rapidly filtered by the kidney and inactivated by rapid cleavage of the N-terminal dipeptide by dipeptidyl peptidase IV (DPP-IV) in vivo, with a half-life of only about two minutes. Therefore, GLP-1 class drug development is mainly developed around long-acting GLP-1 receptor agonists, and a plurality of drugs such as semaglutide, dulaglutide, loxenatide and the like are marketed at present. However, there is an upper limit to the weight-reducing effect of GLP-1 receptor agonists alone, which does not increase with increasing doses, and the gastrointestinal side effects of GLP-1 receptor agonists at high doses are increasingly pronounced. Thus, there remains a need for therapeutic agents that are more safely tolerated, and that are effective in reducing body weight and controlling blood glucose.
Glucose-dependent insulinotropic polypeptide (GIP) is also an incretin in humans, having a similar glucose-dependent insulinotropic effect as GLP-1. In addition, GIP also agonizes GIP receptors in adipose tissues to promote fat metabolism, and GIP also has an appetite-suppressing effect. Studies have shown that the insulinotropic effect of GIP is impaired in diabetics, and therefore GIP receptor agonists have not been developed as hypoglycemic agents in the early days. However, when the GLP-1 receptor agonist and the GIP receptor agonist are combined, the hypoglycemic effect generated by the GLP-1 receptor agonist can fully exert the insulinotropic effect of the GIP receptor agonist, meanwhile, the weight-reducing effect of the GLP-1 receptor agonist can be further enhanced, and finally, excellent hypoglycemic and weight-reducing effects are generated.
GLP-1 receptors and GIP receptors corresponding to GLP-1 and GIP both belong to the GPCR receptor family, have similar protein structures and binding mechanisms, and make it possible to design dual agonists for these two receptors. A dual agonist capable of acting on both GLP-1 receptor and GIP receptor can exert the GLP-1 and GIP activities simultaneously. GLP-1 can reduce blood sugar and suppress appetite; GIP mainly stimulates insulin secretion. The two activities of the GLP-1 receptor and the GIP receptor double agonist are matched with each other, and a feedback mechanism is formed according to the concentration of blood sugar, so that the blood sugar can be controlled, and the weight can be reduced. For the treatment of diabetes, obesity, dual GLP-1/GIP receptor agonists have significant advantages over the GLP-1 analogs alone.
Currently reported dual GLP-1/GIP receptor agonists generally have GIP receptor agonistic activity similar to or stronger than native GIP, and GLP-1 receptor agonistic activity similar to or weaker than native GLP-1. For example, the GIP receptor agonistic activity of the marketed tirzepatide is similar to that of native GIP, and its GLP-1 receptor agonistic activity is about 13-fold weaker than that of native GLP-1.
Disclosure of Invention
The invention provides a long-acting GLP-1/GIP receptor dual agonist and application thereof, wherein the agonist has different GLP-1 receptor and GIP receptor agonistic activity ratios and has strong GLP-1 receptor agonistic activity and weak GIP receptor agonistic activity, thereby improving the hypoglycemic, weight-reducing and lipid-regulating functions of the GLP-1/GIP receptor dual agonist; the preparation method has great potential in preparing medicaments for treating metabolic syndrome, such as diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia and the like.
In order to achieve the above object, the present invention has the following technical scheme:
a long-acting GLP-1/GIP receptor dual agonist, wherein the amino acid sequence of the GLP-1/GIP receptor dual agonist has the general formula:
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Arg-Ala-Xaa 1 -Glu-Xaa 2 -Ile-Ala-Xaa 3 -Arg-Le u-Phe-Ile-Glu-Trp-Leu-Ile-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 wherein: xaa 1 Selected from Met or Leu; xaa 2 Selected from Lys with modified side chains; xaa 3 Selected from Val or Ala;
the side chain modified Lys is selected from
Wherein: n is a natural number, and n is more than or equal to 16 and less than or equal to 20.
Preferably, n is 16, 18 or 20.
Preferably, the sequence structure of the receptor dual agonist is selected from any one of the amino acid sequences shown in SEQ ID NO: 1-2:
SEQ ID NO:1
SEQ ID NO:2
the invention also provides pharmaceutically acceptable salts of the long-acting GLP-1/GIP receptor dual agonists.
The invention also provides a medicament prepared from the long-acting GLP-1/GIP receptor dual agonist, wherein the medicament comprises any one of a tablet, a capsule, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound preparation.
The invention also provides a pharmaceutical composition prepared from the long-acting GLP-1/GIP receptor dual agonist, which comprises the long-acting GLP-1/GIP receptor dual agonist, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprises pharmaceutically acceptable salts, pharmaceutically acceptable carriers or diluents of the long-acting GLP-1/GIP receptor dual agonists.
The invention also provides the application of the long-acting GLP-1/GIP receptor dual agonist or the pharmaceutically acceptable salt of the long-acting GLP-1/GIP receptor dual agonist or the medicament or the pharmaceutical composition in preparing medicaments for treating metabolic diseases or symptoms; the metabolic disease or disorder includes diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.
Compared with the prior art, the invention has the beneficial technical effects that:
(1) The GLP-1/GIP receptor dual agonist provided by the invention has the remarkable weight reduction and weight increase prevention effects while reducing blood sugar more effectively, and can be used for better regulating lipid metabolism;
(2) The N-terminal amino acid His of the GLP-1/GIP receptor dual agonist sequence of the invention is consistent with native GLP-1, without using Tyr commonly used in GLP-1/GIP receptor dual agonist design, which results in better GLP-1 receptor agonistic activity and hypoglycemic activity. In addition, the sequence structure of the GLP-1/GIP receptor dual agonist provided by the invention enables the GLP-1/GIP receptor dual agonist provided by the invention to have strong GLP-1 receptor agonistic activity and weaker GIP receptor agonistic activity, and unexpectedly leads to the GLP-1/GIP receptor dual agonist provided by the invention to have more excellent hypoglycemic, weight-reducing and lipid-regulating effects compared with the GLP-1/GIP receptor dual agonist on the market, thereby bringing about unexpected beneficial effects;
(3) The GLP-1/GIP receptor dual agonist has no agonism activity to the glucagon receptor, can avoid hyperglycemia side effect caused by agonism of the glucagon receptor, and has better action selectivity to the glucagon receptor than the marketed GLP-1/GIP receptor dual agonist;
(4) The GLP-1/GIP receptor dual agonist provided by the invention is stable in chemical property and has pharmacokinetic characteristics for supporting at least once weekly administration; the GLP-1/GIP receptor double agonist provided by the invention has better treatment effect on metabolic diseases such as T2DM, obesity, dyslipidemia and the like than the existing marketed drugs. Therefore, the GLP-1/GIP receptor double agonist provided by the invention is suitable for being used as an active ingredient of medicaments for treating metabolic diseases, such as diabetes, obesity, nonalcoholic fatty liver disease, nonalcoholic fatty hepatitis, dyslipidemia and the like.
Drawings
FIG. 1 shows the acute hypoglycemic effect of each subject of the invention in db/db mice.
Figure 2 shows the percent change in body weight of each subject of the invention over a 21 day period of prolonged dosing in DIO mice.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
Unless defined otherwise herein, scientific and technical terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms and methods used in connection with chemistry, molecular biology, cell biology, pharmacology, according to the invention, are well known and commonly used in the art.
All combinations of the various elements disclosed herein are within the scope of the invention. Furthermore, the scope of the invention should not be limited by the specific disclosure provided below.
Further, the amino acids mentioned in the present invention may be abbreviated as follows according to the naming convention of IUPAC-IUB:
alanine (Ala, a); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); valine (Val, V).
Further, unless explicitly indicated, amino acid residues in the polypeptides of the invention are preferably in the L configuration.
Further, "-NH on the C-terminus of the sequence 2 "part indicates the amide group at the C-terminus-CONH 2 )。
Further, in addition to natural amino acids, unnatural amino acids, alpha-aminoisobutyric acid (Aib) are used in the sequences of the invention.
Furthermore, the polypeptide compound can be synthesized by a polypeptide solid-phase synthesis method or can be produced by a genetic engineering technology.
The following specific embodiments are provided in order to illustrate the present invention in more detail, but the aspects of the present invention are not limited thereto.
Example 1
Synthesis of polypeptide Compound of SEQ ID NO. 1
(1) Swelling of the resin
0.382g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.262mmol/g was weighed into a 25mL reactor, the resin was alternately washed 1 time with 7mL of dichloromethane and methanol, 2 times with 7mL of dichloromethane, then the resin was swollen with 7mL of dichloromethane for 1h, and finally the resin was washed 3 times with 7mL of LDMF.
(2) Removal of Fmoc protecting groups from resin
Transferring the swelled resin into a PSI-200 semiautomatic polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) for reaction at room temperature for 30min, filtering out the deprotected solution, and cleaning the resin for 4 times by 7mL of LDMF for 1.5min each time to obtain Rink resin with Fmoc protecting groups removed.
(3) Synthesis of Fmoc-Ser-Rink amide-MBHAresin
Fmoc-Ser (tBu) -OH (0.4 mmol) was weighed, dissolved in 3mL of DMF, 2mL of DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, pre-activated for 30min, the activated amino acid was added to the reactor, the reaction was stirred at room temperature for 2h, the reaction solution was filtered off, the resin was washed with 7mL of MF for 4 times, and Kaiser reagent was used to detect if the reaction coupling was complete, and if not, 2 times.
(4) Extension of peptide chain
And (3) according to the sequence of the peptide chain, repeating the deprotection and coupling steps to sequentially connect corresponding amino acids until the peptide chain is synthesized. The Lys site in which the side chain at position 16 is modified employs Fmoc-Lys (Dde) -OH protection strategy, while the N-terminal His employs Boc-His (Boc) -OH.
(5) Modification of Lys side chains
After the peptide chain synthesis is completed, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove Dde protecting group of 16-position Lys, after the Dde protecting group is removed, 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt are added, and the oscillation condensation reaction is carried out for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-AEEA-OH,0.4mmol DIC and 0.44mmol HOBt were added again and the reaction was performed by shaking for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-Glu-OtBu,0.4mmol DIC and 0.44mmol HOBt were added and the reaction was performed by shaking for 2h. After Fmoc protecting groups were removed, 0.4mmol of mono-tert-butyl octadecanedioate, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2h, and after the reaction was completed, the resin was washed 4 times with 7 mM LDMF.
(6) Cleavage of polypeptides
The obtained resin connected with the polypeptide is transferred into a round bottom bottle, 8mL of a cutting agent (TFA/benzyl sulfide/phenol/EDT, 90:5:3:2, V/V) is used for cutting the resin, the resin is subjected to oscillation reaction for 2 hours at room temperature, the cutting solution is poured into 40mL of glacial ethyl ether, the crude product is washed 3 times with 15mL of glacial ethyl ether after refrigerated centrifugation, and finally the crude peptide is obtained by drying with nitrogen.
(7) Purification of polypeptides
Dissolving the target polypeptide crude product in water, filtering with 0.25 μm microporous membrane, and purifying with Shimadzu semi-preparative reversed phase HPLC system. Chromatographic conditions are C18 reverse phase preparation column; mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol; the flow rate is 6mL/min; the detection wavelength was 214nm. Eluting with linear gradient (30% B-80% B/50 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity of 0.11g or more than 99%, and confirming molecular weight of target polypeptide by mass spectrometry. The theoretical relative molecular mass is 4912.6.ESI-MS M/z calculated [ M+3H] 3+ 1638.5,[M+4H] 4+ 1229.2; observed value [ M+3H] 3+ 1638.2,[M+4H] 4+ 1228.9。
Example 2
Synthesis of polypeptide compound of SEQ ID No. 2
The synthesis method is the same as that of example 1, 0.12g of a pure product is obtained by collecting target peaks and freeze-drying, the purity is more than 99%, and the molecular weight of target polypeptides is confirmed by mass spectrometry. The theoretical relative molecular mass is 4866.6.ESI-MS M/z calculated [ M+3H] 3+ 1623.2,[M+4H] 4+ 1217.7; observed value [ M+3H] 3+ 1622.9,[M+4H] 4+ 1217.4。
Example 3
Determination of agonistic Activity of polypeptide Compounds on GLP-1 receptor, GIP receptor and Glucoago receptor
Agonism of the receptor by the polypeptide compounds is determined by a functional assay that measures cAMP response of HEK-293 cell lines stably expressing human GLP-1 receptor, GIP receptor or glucagon receptor. HEK-293 cell lines stably expressing GLP-1 receptor, GIP receptor or glucoon receptor were cultured in DMEM+100. Mu.g/mL Hygromycin B medium containing 10% fetal bovine serum at 37℃and carbon dioxide concentration of 5%. Cell passage: the old medium was removed and washed once with PBS, then 1mL Cell Dissociation Buffer solution was added and incubated at 37℃for about 2 min. When the cells were detached from the bottom of the dish, about 5ml of complete medium, pre-warmed at 37℃was added. The cell suspension was gently swirled with a pipette to separate the aggregated cells. The cell suspension was transferred to a sterile centrifuge tube and centrifuged at 1000rpm for 5min to collect the cells for experimental or subculture. To maintain the physiological activity of the cells, the degree of fusion of the experimental cells was controlled to be about 80%. Next, 1 x Stimulation Buffer was prepared for use according to the kit instructions; gradient dilution of polypeptide compounds with DMSO followed by 10-fold gradient dilution of compounds with 1 x Stimulation Buffer; culturing stable transfer line cells to 80% fusion; cell Dissociation Buffer solution digestion treatment cells were collected and counted and inoculated with 9. Mu.L/well in 384-well plates. 1 μl of the diluted compound was added to the corresponding experimental well, centrifuged and incubated at 37deg.C for 30min. Eu-cAMP is usedThe Detection buffer was diluted to the working concentration, and 5. Mu.L/well was added to the corresponding experimental well. Diluting ULIght-anti-cAMP to working concentration by using a Detection buffer, and then adding 5 mu L/well into a corresponding experimental hole; after centrifugation, the mixture was left at room temperature for 1h. After incubation, 665nm and 620nm reads were detected using a Perkin Elmer multifunctional microplate reader. Curve fitting and EC were performed by GraphPad Prism software nonlinear regression method by plotting Ratio (665/620) versus compound concentration 50 And (5) calculating.
The test data (nM) in the examples of the present application are shown in Table 1 below, and although the test data is stated in terms of a number of significant digits, it should not be considered to indicate that the data has been determined to be exactly a significant digit.
Table 1: EC of polypeptide compounds to human GLP-1 receptor, GIP receptor and glucagon receptor 50 Value (expressed in nM)
As shown in Table 1, the agonistic activity of the polypeptide compound of the present invention to GLP-1 receptor is stronger than that of natural GLP-1 and tirzepatide, the agonistic activity of the polypeptide compound of the present invention to GIP receptor is weaker than that of natural GIP (about 45-160 times weaker) and tirzepatide (about 40-141 times weaker), indicating that the polypeptide compound of the present invention has strong GLP-1 receptor agonistic activity and weaker GIP receptor agonistic activity, and the ratio of agonistic activity to GLP-1 receptor and GIP receptor is different from tirzepatide, and also conforms to the characteristics of dual agonist of the present invention. In addition, the agonistic activity of the polypeptide compound of the present invention to the glucagon receptor is lower than that of tirzepatide, which indicates that the polypeptide compound of the present invention has better selectivity of the action of the glucagon receptor, so that the hyperglycemia side effect caused by agonizing the glucagon receptor is not generated.
Example 4
Pharmacokinetic properties of polypeptide Compounds in SD rats
Male SD rats (200-250 g) were randomly grouped, 3 animals per group, fasted overnight prior to the experiment, and then each group of rats was given 50nmol/kg of semaglutinide subcutaneously, SEQ ID NO:1, SEQ ID NO:2. Blood samples were collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h and 48h post-dose. After precipitation of the proteins using acetonitrile, plasma samples were analyzed by LC-MS. The in vivo half-life of each test article was calculated using WinnLin 5.2.1 (non-compartmental model) (Table 2).
Table 2: half-life of polypeptide compounds in rats
| Sample of | T 1/2 (h) |
| Semaglutide | 9.6 |
| SEQ ID NO:1 | 11.6 |
| SEQ ID NO:2 | 10.5 |
As the results in table 2 show, the in vivo half-life of the polypeptide compounds of the present invention is significantly prolonged over once a week dosing semaglutine already on the market, demonstrating that the polypeptide compounds of the present invention have pharmacokinetic profiles supporting at least once a week dosing.
Example 5
Acute hypoglycemic effects of polypeptide compounds in db/db mice
Male db/db mice (8 weeks old), randomized, 6 per group. After 7 days of adaptive feeding, each group of mice was fasted for 12 hours, and then each group of mice was subcutaneously administered physiological saline (saline, blank group, 10 mL/kg), semaglutinide (30 nmol/kg), tirzepatide (30 nmol/kg), SEQ ID NO:1 (30 nmol/kg), SEQ ID NO:2 (30 nmol/kg), respectively. After administration for 60 minutes, each group of mice was orally administered glucose solution (1.5 g/kg) through a stomach-filling needle, and blood glucose levels of each group of mice were measured with a blood glucose meter at the following time points, -60min, 0min, 15min, 30min, 60min, 120 min.
As shown in the results of FIG. 1, the polypeptide compound of the present invention has excellent acute hypoglycemic effect in db/db mice, and the acute hypoglycemic effect of SEQ ID NO. 1 and SEQ ID NO. 2 is also superior to semaglutide and tirzepartial.
Example 6
Influence of polypeptide Compounds on weight and blood lipid in diet-induced obese (DIO) mice
Male C57BL/6J mice, weighing about 22g, were kept on D12492 high fat diet from Research Diets for about 18 weeks to make DIO mouse models (mice weighing >48 g). Before the start of the administration, DIO mice of each group were randomly grouped according to body weight, and 6 groups were physiological saline group (saline, blank control group), positive control group (semaglutide and tirzepatide), and test sample group (SEQ ID NO:1 and SEQ ID NO: 2), respectively. Each group of mice was subcutaneously injected with normal saline (10 mL/kg), semaglutide (30 nmol/kg), tirzepatide (30 nmol/kg), SEQ ID NO:1 (30 nmol/kg), SEQ ID NO:2 (30 nmol/kg), and the administration period was 21 days every two days. The mice weight change was recorded daily and Nuclear Magnetic Resonance (NMR) was used to measure the body fat mass of each group of mice before and at the end of the experiment. At the end of the experiment, each group of mice was sacrificed, liver tissue was taken to measure liver Triglyceride (TG) and Total Cholesterol (TC) content, blood serum was taken at the same time, and each group of mice serum glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), triglyceride (TG) and Total Cholesterol (TC) content was measured.
Table 3: body weight and body fat changes in DIO mice over a 3 week dosing period
| Sample (dose) | Overall weight change (%) | Body fat change (%) |
| Blank control (normal saline group) | -0.6±1.0 | 0.3±0.1 |
| Semaglutide(30nmol/kg) | -19.7±3.2 *** | -23.6±3.8 *** |
| Tirzepatide(30nmol/kg) | -31.4±3.7 *** | -39.6±5.9 *** |
| SEQ ID NO:1(30nmol/kg) | -41.0±1.7 ***,### | -50.4±4.2 ***,### |
| SEQ ID NO:2(30nmol/kg) | -38.3±2.0 ***,### | -46.8±3.2 ***,### |
*** : p compared with the blank control group<0.001； ### : p compared to semaglutide and tirzepatide groups<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in the results of FIG. 2 and Table 3, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention can significantly reduce the body weight and body fat content of mice by continuous administration in DIO mice for 3 weeks, and the effect of the polypeptide compounds of the present invention in reducing the body weight and body fat is significantly stronger than that of positive control drugs semaglutide and tirzenotice.
Notably, the polypeptide compounds of the present invention SEQ ID NO. 1 and SEQ ID NO. 2 differ in the ratio of agonistic activity at the GLP-1 receptor and the GIP receptor from tirzepatide. The agonistic activity of Tirzepatide to GLP-1 receptor is about 13 times weaker than natural GLP-1, the agonistic activity to GIP receptor is similar to natural GIP, and the agonistic activity to GLP-1 receptor of the polypeptide compound SEQ ID NO 1 of the present invention is about 2.4 times stronger than natural GLP-1, the agonistic activity to GIP receptor is about 45 times lower than natural GIP; the polypeptide compound SEQ ID NO. 2 has similar agonistic activity to GLP-1 receptor as natural GLP-1 and about 160 times lower agonistic activity to GIP receptor than natural GIP. However, the strong GLP-1 receptor agonistic activity and the weak GIP receptor agonistic activity of the polypeptide compound of the invention bring about better weight-saving and body fat-reducing effects instead.
Table 4: liver Triglyceride (TG) and Total Cholesterol (TC) levels 3 weeks after DIO mice treatment
| Sample (dose) | Total cholesterol (mg/g) | Triglyceride (mg/g) |
| Blank control (normal saline group) | 11.4±1.2 | 96.5±8.1 |
| Semaglutide(30nmol/kg) | 8.9±0.8 *** | 70.5±5.9 *** |
| Tirzepatide(30nmol/kg) | 7.5±0.5 *** | 62.2±3.8 *** |
| SEQ ID NO:1(30nmol/kg) | 4.7±0.3 ***,### | 41.1±2.7 ***,### |
| SEQ ID NO:2(30nmol/kg) | 4.9±0.4 ***,### | 44.8±4.3 ***,### |
*** : p compared with the blank control group<0.001； ### : p compared to semaglutide and tirzepatide groups<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
Table 5: serum glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) levels after 3 weeks of DIO mice treatment
| Sample (dose) | Glutamic pyruvic transaminase (U/L) | Glutamic-oxaloacetic transaminase (U/L) |
| Blank control (normal saline group) | 641±98 | 603±41 |
| Semaglutide(30nmol/kg) | 338±51 *** | 319±29 *** |
| Tirzepatide(30nmol/kg) | 297±24 *** | 266±22 *** |
| SEQ ID NO:1(30nmol/kg) | 143±12 ***,### | 126±15 ***,### |
| SEQ ID NO:2(30nmol/kg) | 138±28 ***,### | 133±14 ***,### |
*** : p compared with the blank control group<0.001； ### : p compared to semaglutide and tirzepatide groups<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in tables 4 and 5, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 prepared in the embodiment of the invention can be continuously administered in DIO mice for 3 weeks, can obviously reduce the liver triglyceride and total cholesterol content of the mice, can obviously reduce the serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase content of the mice, and have the effect obviously stronger than that of positive control drugs semaglutinide and tirzepatide, thus showing that the polypeptide compound has good prospect for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.
Table 6: serum Triglyceride (TG) and Total Cholesterol (TC) levels 3 weeks after DIO mice treatment
| Sample (dose) | Total cholesterol (mmol/L) | Triglyceride (mmol/L) |
| Blank control (normal saline group) | 11.1±1.2 | 1.9±0.2 |
| Semaglutide(30nmol/kg) | 8.2±0.6 *** | 1.1±0.1 *** |
| Tirzepatide(30nmol/kg) | 7.0±0.4 *** | 1.0±0.2 *** |
| SEQ ID NO:1(30nmol/kg) | 5.1±0.3 ***,### | 0.5±0.1 ***,### |
| SEQ ID NO:2(30nmol/kg) | 5.3±0.2 ***,### | 0.6±0.1 ***,### |
*** : p compared with the blank control group<0.001； ### : p compared to semaglutide and tirzepatide groups<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown by the results in Table 6, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention can significantly reduce serum triglyceride and total cholesterol contents of mice by continuously administering in DIO mice for 3 weeks, and the effect of the polypeptide compounds of the present invention for reducing serum lipid (triglyceride and total cholesterol) contents is significantly stronger than that of positive control drugs semaglutide and tirzepatide.
Example 7
Effect of polypeptide Compounds on db/db mouse glycosylated hemoglobin (HbA 1 c) and blood glucose
Male db/db mice (8 weeks old), randomized, 6 per group. Physiological saline group (blank control group), positive control group (semaglutide and tirzepatide) and test sample group (SEQ ID NO:1 and SEQ ID NO: 2), respectively. After one week of adaptive feeding, the initial HbA1c (%) values and fasting blood glucose values were measured before the start of the blood-taking measurement treatment. Each group of mice was subcutaneously injected with normal saline (10 mL/kg), semaglutide (30 nmol/kg), tirzepatide (30 nmol/kg), SEQ ID NO:1 (30 nmol/kg), SEQ ID NO:2 (30 nmol/kg), and the administration period was 35 days every two days. The mice were fasted overnight after the end of treatment and blood was taken to measure the fasting blood glucose values and HbA1c (%).
Table 7: hbA1c (%) change in db/db mice over a 35 day dosing period
| Sample (dose) | HbA1c% (before treatment) | HbA1c% (after treatment) |
| Blank control (normal saline group) | 6.4±0.3 | 7.6±0.5 |
| Semaglutide(30nmol/kg) | 6.4±0.2 | 6.2±0.2 *** |
| Tirzepatide(30nmol/kg) | 6.5±0.3 | 6.0±0.3 *** |
| SEQ ID NO:1(30nmol/kg) | 6.5±0.4 | 5.2±0.2 ***,### |
| SEQ ID NO:2(30nmol/kg) | 6.6±0.3 | 5.3±0.3 ***,### |
*** : p compared with the blank control group<0.001； ### : p compared to semaglutide and tirzepatide groups<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in the results of Table 7, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention can significantly reduce HbA1c values of mice after 35 days of continuous administration in db/db mice, and HbA1c values of mice in the group of the polypeptide compounds of the present invention after treatment are significantly lower than those of positive controls semaglutide and tirzepatide, indicating that the polypeptide compounds of the present invention have excellent glycemic control.
Table 8: fasting blood glucose changes in db/db mice over a 35 day dosing period
| Sample (dose) | Fasting blood glucose change (%) |
| Blank control (normal saline group) | +3.5±0.2％ |
| Semaglutide(30nmol/kg) | -2.3±0.2％ *** |
| Tirzepatide(30nmol/kg) | -4.1±0.5％ *** |
| SEQ ID NO:1(30nmol/kg) | -9.5±0.4％ ***,### |
| SEQ ID NO:2(30nmol/kg) | -9.2±0.6％ ***,### |
*** : p compared with the blank control group<0.001； ### : p compared to semaglutide and tirzepatide groups<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in the results of Table 8, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 prepared in the examples of the present invention can significantly reduce the fasting blood glucose value of db/db mice by continuous administration in db/db mice for 35 days, indicating that the polypeptide compounds of the present invention have excellent blood glucose control effect, and the blood glucose control effect of the polypeptide compounds of the present invention is significantly stronger than that of the positive control drugs semaglutide and tirzepatide.
Claims (5)
1. A long-acting GLP-1/GIP receptor dual agonist is characterized in that the sequence structure of the receptor dual agonist is selected from any one of amino acid sequences shown as SEQ ID NO. 1-2:
SEQ ID NO:1
SEQ ID NO:2
2. a class of pharmaceutically acceptable salts of the long acting GLP-1/GIP receptor dual agonists according to claim 1.
3. A pharmaceutical formulation prepared from the long acting GLP-1/GIP receptor dual agonist of claim 1, wherein the pharmaceutical formulation comprises any one of a tablet, capsule, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound formulation.
4. A pharmaceutical composition prepared from a class of long-acting GLP-1/GIP receptor dual agonists, characterized in that the pharmaceutical composition comprises a class of long-acting GLP-1/GIP receptor dual agonists according to claim 1, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprises a pharmaceutically acceptable salt, a pharmaceutically acceptable carrier or diluent of a class of long acting GLP-1/GIP receptor dual agonists as claimed in claim 2.
5. Use of a class of long acting GLP-1/GIP receptor dual agonists according to claim 1 or a class of long acting GLP-1/GIP receptor dual agonists pharmaceutically acceptable salts according to claim 4 or a class of agents according to claim 3 or a pharmaceutical composition according to claim 4 for the preparation of a medicament for the treatment of a metabolic disease or disorder; the metabolic disease or disorder includes diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.
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| WO2016043533A1 (en) * | 2014-09-16 | 2016-03-24 | Hanmi Pharm. Co., Ltd. | Use of a long acting glp-1/glucagon receptor dual agonist for the treatment of non-alcoholic fatty liver disease |
| CN111253475A (en) * | 2020-02-18 | 2020-06-09 | 江苏诺泰澳赛诺生物制药股份有限公司 | GLP-1 agonist polypeptide compound and salt thereof, and synthesis method and application thereof |
| CN114349828A (en) * | 2020-11-27 | 2022-04-15 | 江苏师范大学 | GLP-1/glucagon receptor dual agonist and application thereof |
| CN114437181A (en) * | 2022-01-25 | 2022-05-06 | 深圳深创生物药业有限公司 | GLP-1R/GCGR/GIPR triple receptor agonist and application thereof |
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| WO2016043533A1 (en) * | 2014-09-16 | 2016-03-24 | Hanmi Pharm. Co., Ltd. | Use of a long acting glp-1/glucagon receptor dual agonist for the treatment of non-alcoholic fatty liver disease |
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| CN114349828A (en) * | 2020-11-27 | 2022-04-15 | 江苏师范大学 | GLP-1/glucagon receptor dual agonist and application thereof |
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