CN117603364A - A class of GLP-1/glucagon/Y2 receptor triple agonists and their applications - Google Patents

Abstract

The invention provides a type of GLP-1/glucagon/Y ₂ receptor triple agonist and its application. The triple agonist has triple agonist activity on human GLP-1, glucagon and Y ₂ receptors. The GLP‑1/glucagon/Y ₂ receptor triple agonist of the present invention can effectively reduce blood sugar and at the same time have the beneficial effects of significantly reducing weight loss and treating NAFLD. The GLP‑1/glucagon/Y ₂ receptor triple agonist provided by the present invention has stable chemical properties, has pharmacokinetic characteristics that support once-a-day administration, and is suitable for the treatment of metabolic diseases (such as diabetes, obesity, non-alcoholic fatty liver disease, dyslipidemia, etc.).

Description

A class of GLP-1/glucagon/Y2 receptor triple agonists and their applications

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to a type of GLP-1/glucagon/Y ₂ receptor triple agonist and its application.

Background technique

Obesity and its related metabolic syndrome have become a global public health problem. Many metabolic syndromes such as type 2 diabetes (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and abnormal lipid metabolism The incidence and course of the disease are closely related to obesity. GLP-1 is a glucose-dependent hypoglycemic peptide hormone secreted by small intestinal L cells. Its main function is to promote the secretion of insulin. Glucagon glycopeptide-1 (GLP-1) can suppress appetite and delay gastric emptying to reduce weight. Although GLP-1 has excellent hypoglycemic effect and certain weight loss effect, if you need to achieve better weight loss effect, you generally need to increase the dosage, and large doses of GLP-1 drugs are prone to gastrointestinal tract complications. Side effects and poor tolerability lead to a narrow therapeutic window. Therefore, there remains a need for more safe and tolerable therapeutic agents that are effective for weight loss and glycemic control.

Glucagon is a hormone produced in the α-cells of the pancreas. It acts on the liver under stress conditions such as cold and hunger to decompose glycogen in the liver and increase blood sugar. In addition, glucagon also has the effects of promoting lipolysis, fat oxidation, and fever in the body (Diabetologia, 2017, 60, 1851-1861). Long-term administration can show weight loss effects by increasing energy metabolism, but these effects of glucagon Beneficial effects on energy metabolism are prevented by its inherent glycemic effects. Peripheral administration of GLP-1 and glucagon in animals and humans can achieve simultaneous hypoglycemic and weight loss effects. There are currently a variety of GLP-1/glucagon receptor dual agonists in the clinical research stage, but GLP-1/glucagon receptor Dual agonists have the disadvantage of greater gastrointestinal side effects.

Neuropeptide Y (NPY) is a 36 amino acid peptide neurotransmitter, a member of the pancreatic polypeptide class of neurotransmitters/neurohormones that has been shown to be present in both the peripheral and central nervous systems. NPY is one of the most potent appetizers known and has been shown to play an important role in regulating food intake in animals, including humans. There are four subtypes of NPY receptors, Y ₁ , Y ₂ , Y ₄ and Y ₅ . Among them, neuropeptide-2 (Y ₂ ) receptor is widely distributed in the central nervous system of rodents and humans. In the hypothalamus, _Y2 mRNA is localized in the arcuate nucleus, preoptic nucleus, and dorsomedial nucleus. In the human brain, _Y2 receptors are the major NPY receptor subtype. In the arcuate nucleus, more than 80% of NPY neurons co-express _Y2 receptor mRNA. Choosing to activate _Y2 receptors can inhibit food intake and have the effect of weight loss.

Peptide YY _3-36 (PYY _3-36 ) is a 34-amino-acid linear peptide that has Y ₂ receptor agonistic activity. It has good reduction in GLP-1 and PYY _3-36 combined injection in animals and humans. Glucose and weight loss effects, indicating that stimulating GLP-1 and Y ₂ receptors at the same time can further utilize the appetite suppressive effect brought about by stimulating Y ₂ receptors to produce better weight loss while maintaining the hypoglycemic effect of GLP-1. Heavy role. Novo Nordisk reported in 2021 a class of hybrid peptides of GLP-1 and short-chain PYY _3-36 analogs with dual agonistic activities at GLP-1 and _Y2 receptors. However, this type of compound has not been modified for long-acting, and its in vivo stability is relatively limited, making it impossible to achieve long-acting administration (Angew. Chem. Int. Ed. Engl., 2021, 6; 60 (15): 8268-8275) . Brandon et al. revealed a type of hybrid peptide of exendin-4 and short-chain PYY _3-36 analogues, which has good dual agonistic activity at GLP-1 and Y ₂ receptors, similar to the compounds reported by Novo Nordisk. The compound has not undergone any long-lasting modification and has poor stability (J. Med. Chem., 202, 28; 64(2): 1127-1138).

At present, there is still a lack of GLP-1/glucagon/Y ₂ receptor triple agonists that can simultaneously integrate the beneficial effects of GLP-1, glucagon and PYY _3-36 on blood sugar, food intake and energy metabolism, so as to develop a more active anti-metabolic disease drugs.

Contents of the invention

The object of the present invention is to provide a kind of GLP-1/glucagon/Y ₂ receptor triple agonist and its application. The agonist can have triple agonist activity and stability on human GLP-1 receptor, glucagon and Y ₂ receptor. It is highly toxic and has the pharmacokinetic characteristics of once-a-day dosing; it has greater potential to be used in the preparation of drugs for the treatment of metabolic syndrome, such as diabetes, obesity, non-alcoholic fatty liver disease, dyslipidemia and other diseases.

In order to achieve the above-mentioned object of the invention, the technical solutions of the present invention are as follows:

A type of GLP-1/glucagon/Y ₂ receptor triple agonist, the amino acid sequence of the GLP-1/glucagon/Y ₂ receptor triple agonist is one of the following sequences:

(1)SEQ ID NO:1

(2)SEQ ID NO:2

(3)SEQ ID NO:3

(4)SEQ ID NO:4

(5)SEQ ID NO:5

(6)SEQ ID NO:6

(7)SEQ ID NO:7

(8)SEQ ID NO:8

(9)SEQ ID NO:9

The present invention also provides pharmaceutically acceptable salts of GLP-1/glucagon/Y ₂ receptor triple agonists.

Preferably, the salt is a salt formed by a GLP-1/glucagon/Y ₂ receptor triple agonist and one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, Oxalic acid, lactic acid, succinic acid.

The present invention also provides pharmaceutical preparations prepared from GLP-1/glucagon/Y ₂ receptor triple agonists. The pharmaceutical preparations are tablets, capsules, inhalants, sprays, injections, Films, patches, emulsions, suppositories or compound preparations are composed of a type of GLP-1/glucagon/Y ₂ receptor triple agonist and pharmaceutically acceptable pharmaceutical excipients, carriers or diluents.

The present invention also provides a pharmaceutical composition containing a type of GLP-1/glucagon/Y ₂ receptor triple agonist, which pharmaceutical composition uses the above GLP-1/glucagon/Y ₂ receptor triple agonist as an effective raw material, or Its pharmaceutically acceptable salt is an effective raw material, plus a pharmaceutically acceptable carrier or diluent.

The present invention also provides the GLP-1/glucagon/Y ₂ receptor triple agonist of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof or a medicament thereof for use in the treatment of metabolic diseases or disorders. uses in medicines. In particular aspects, the metabolic disease or condition is diabetes, obesity, NAFLD, dyslipidemia. In certain aspects, diabetes is T1DM, T2DM or gestational diabetes. In certain aspects, the medicament is used to treat more than one metabolic disease or condition, for example, diabetes and obesity; diabetes and NAFLD; diabetes and dyslipidemia; diabetes, dyslipidemia, and obesity.

Compared with the existing technology, the beneficial effects of the present invention are:

The present invention provides a class of variants designed based on GLP-1 analogues, glucagon and PYY _3-36 sequences, which retain the therapeutic effect of GLP-1 analogues on diabetes while having the beneficial effects of glucagon on metabolism and PYY _3-36 on appetite. It can effectively reduce blood sugar and significantly reduce weight loss and treat NAFLD. In addition, the GLP-1/glucagon/Y ₂ receptor triple agonist provided by the present invention has stable chemical properties and has pharmacokinetic characteristics that support once-a-day administration. The triple agonist provided by the present invention has a better therapeutic effect on metabolic diseases such as T2DM, obesity, NAFLD, and dyslipidemia than existing marketed drugs. Therefore, the triple agonist provided by the present invention is suitable as an active ingredient in drugs for treating metabolic diseases (such as diabetes, obesity, NAFLD, dyslipidemia, etc.).

Description of drawings

Figure 1 shows a schematic diagram of the results of a single administration of each test substance on feeding inhibition in ICR mice;

Figure 2 shows a schematic diagram of the results of the acute hypoglycemic effect of a single administration of each test substance on ICR mice;

Figure 3 shows a schematic diagram of the percentage change in body weight of each test substance in DIO mice after long-term administration for 21 days.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meaning commonly understood by one of ordinary skill in the art. In general, the terms and methods used in connection with chemistry, biology, and pharmacology described herein are well known and commonly used in the art.

In addition, the abbreviations of the amino acids involved in the present invention according to the IUPAC-IUB naming rules are as follows:

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).

In addition, unless explicitly stated, all amino acid residues in the polypeptide compounds of the present invention are preferably in the L configuration.

Additionally, the " _-NH2 " portion on the C-terminus of the sequence indicates the amide group ( _-CONH2 ) on the C-terminus.

In addition, in addition to natural amino acids, the sequence of the present invention also uses the unnatural amino acid d-serine ( _dSer , _dS ).

Example 1

Synthesis of polypeptide compounds of SEQ ID NO:1

(1) Swelling of resin

Weigh 0.278g (0.1mmol equivalent) of RinkAmide MBHA resin with a loading capacity of 0.36mmol/g, put it into a 25mL reactor, wash the resin once with 7mL of DCM and methanol alternately, and wash the resin with 7mL of DCM twice. Then the resin was swollen with 7 mL of DCM for 1 h, and finally the resin was washed with 7 mL of DMF three times.

(2) Removal of resin Fmoc protective group

Transfer the swollen resin to the PSI-200 peptide synthesizer, add 7mL of 20% piperidine/DMF (v/v) and react at room temperature for 5 minutes. Filter out the deprotection solution, wash the resin once with 7mL of DMF, and then add 7mL of 20% piperidine/DMF. DMF (v/v) deprotection solvent reacted with the resin for 15 minutes, and finally washed the resin 4 times with 7 mL of DMF for 2 minutes each time to obtain the Rink resin with the Fmoc protecting group removed.

(3)Synthesis of Fmoc-Tyr-Rink amide-MBHAResin

Weigh Fmoc-Tyr(tBu)-OH (0.4mmol), dissolve it in 2mL DMF, add 3mLDIC/HOBt (0.4mmol/0.44mmol) condensing agent, add it to the reactor, shake at room temperature for 2h, filter the reaction solution and use 7mL Wash the resin 4 times with DMF, and use Kaiser reagent to check whether the reaction coupling is complete. If it is incomplete, couple it twice.

(4) Extension of the peptide chain in the PYY part (right part)

According to the sequence of the PYY partial peptide chain (IKPEAPGEDA SPEELNRYYA SLRHY LNLVT RQRY-NH ₂ ), repeat the above deprotection and coupling steps to connect the corresponding amino acids in sequence until the peptide chain is synthesized.

(5)Synthesis of connecting arms

After the synthesis of the PYY partial peptide chain is completed, continue to synthesize the connecting arm part, add 0.4mmol Fmoc-AEEA-OH, 0.4mmol DIC and 0.44mmol HOBt, and shake the condensation reaction for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt were added again, and the condensation reaction was carried out with shaking for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of 3-maleimidopropionic acid, 0.4 mmol of DIC and 0.44 mmol of HOBt were added, and the condensation reaction was carried out with shaking for 2 hours.

(6) Cleavage of the PYY part containing the connecting arm

Transfer the polypeptide-connected resin obtained above to a round-bottomed bottle, use 5 mL of cutting agent Reagent R (TFA/anisole/phenol/EDT, 90:5:3:2, V/V) to cut the resin, and then The reaction was carried out in an oil bath at a constant temperature of 30°C for 2 hours. The cutting solution was poured into 40 mL of glacial ether. After refrigeration and centrifugation, the crude product was washed three times with 15 mL of glacial ether and finally dried with nitrogen to obtain a crude peptide containing the PYY part of the connecting arm.

(7) Synthesis of GLP-1/glucagon part (left part) sequence

Weigh 0.278g (0.1mmol equivalent) of RinkAmide MBHA resin with a loading capacity of 0.36mmol/g, put it into a 25mL reactor, wash the resin once with 10mL of DCM and methanol alternately, and wash the resin with 10mL of DCM twice. Then the resin was swelled with 10 mL of DCM for 1 h, and finally the resin was washed with 10 mL of DMF three times. Transfer the swollen resin to the PSI-200 peptide synthesizer, add 10 mL of 20% piperidine/DMF (v/v) and react at room temperature for 5 minutes. Filter out the deprotection solution, wash the resin once with 10 mL of DMF, and then add 10 mL of 20% piperidine/ The DMF (v/v) deprotection solvent reacted with the resin for 15 minutes, and finally the resin was washed 4 times with 10 mL of DMF, 1.5 minutes each time, to obtain the Rink resin with the Fmoc protective group removed. Weigh Fmoc-Cys(Trt)-OH (0.4mmol), dissolve it in 2mL of 10% DMF/DMSO (v/v), add 3mL of DIC/HOBt (0.4mmol/0.44mmol) condensing agent, add it to the reactor, and shake at room temperature for reaction. 2h, filter out the reaction solution and wash the resin 4 times with 10 mL DMF. Use Kaiser reagent to check whether the reaction coupling is complete. If it is incomplete, couple it twice.

(8) Extension of the partial peptide chain of GLP-1/glucagon

According to the sequence of the peptide chain, repeat the above deprotection and coupling steps to connect the corresponding amino acids in sequence until the synthesis of the peptide chain is completed. The Lys at the side chain modification site adopts the Fmoc-Lys(Dde)-OH protection strategy, while the N-terminal His uses Boc-His(Boc)-OH.

(9) Modification of partial Lys side chain of GLP-1/glucagon

After the synthesis of the peptide chain is completed, add 7 mL of 2% hydrazine hydrate/DMF (v/v) to selectively remove the Dde protecting group of Lys. After removing the Dde protecting group, add 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44mmol HOBt, shake reaction for 2h. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt were added again, and the condensation reaction was carried out with shaking for 2 h. After removing the Fmoc protecting group, add 0.4 mmol palmitic acid, 0.4 mmol DIC and 0.44 mmol HOBt for condensation reaction for 2 hours. After the reaction is complete, the resin is washed 4 times with 7 mL DMF.

(10) Cleavage of GLP-1/glucagon part

Transfer the polypeptide-connected resin obtained above to a round-bottomed bottle, use 5 mL of cutting agent Reagent R (TFA/anisole/phenol/EDT, 90:5:3:2, V/V) to cut the resin, and then The reaction was carried out in an oil bath at a constant temperature of 30°C for 2 hours. The cutting solution was poured into 40 mL of glacial ether. After refrigeration and centrifugation, the crude product was washed three times with 15 mL of glacial ether and finally dried with nitrogen to obtain the crude peptide of the GLP-1/glucagon part.

(11)Synthesis of target peptides

Dissolve 0.05 mmol of the crude peptide containing the PYY part of the connecting arm and 0.05 mmol of the crude peptide of the GLP-1/glucagon part in 2 mL of NMP, add 0.005 mmol of DIPEA for catalysis, and after 20 minutes of reaction, add 5 mL of 50% Methanol/water (0.1% TFA) was used to stop the reaction, and the reaction solution was filtered with a 0.25 μm microporous membrane and purified by a Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions are C18 reversed-phase preparative column (250mm×20mm, 12μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate is 8mL/min; detection wavelength is 214nm. Use a linear gradient (20% B to 90% B/30 min) to elute, collect the target peak, remove methanol and freeze-dry to obtain 0.012g of pure product, with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9242.3. ESI-MS m/z: calculated values [M+8H] ⁸⁺ 1156.3, [M+9H] ⁹⁺ 1027.9; observed values [M+8H] ⁸⁺ 1156.0, [M+9H] ⁹⁺ 1027.7.

Example 2

Synthesis of SEQ ID NO:2 polypeptide compounds

The synthesis method is the same as in Example 1. The target peak is collected and lyophilized to obtain 0.014g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9532.7. ESI-MS m/z: calculated values [M+8H] ⁸⁺ 1192.6, [M+9H] ⁹⁺ 1060.2; observed values [M+8H] ⁸⁺ 1192.3, [M+9H] ⁹⁺ 1059.9.

Example 3

Synthesis of SEQ ID NO:3 polypeptide compounds

The synthesis method is the same as in Example 1. The target peak is collected and freeze-dried to obtain 0.016g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10113.2. ESI-MS m/z: calculated value [M+9H] ⁹⁺ 1124.7, [M+10H] ¹⁰⁺ 1012.3; observed value [M+9H] ⁹⁺ 1124.6, [M+10H] ¹⁰⁺ 1012.2.

Example 4

Synthesis of polypeptide compounds of SEQ ID NO:4

The synthesis method is the same as in Example 1. The target peak is collected and lyophilized to obtain 0.011g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9413.4. ESI-MS m/z: calculated values [M+7H] ⁷⁺ 1345.8, [M+8H] ⁸⁺ 1177.7; observed values [M+7H] ⁷⁺ 1345.4, [M+8H] ⁸⁺ 1177.3.

Example 5

Synthesis of SEQ ID NO:5 polypeptide compounds

The synthesis method is the same as in Example 1. The target peak is collected and lyophilized to obtain 0.015g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9703.8. ESI-MS m/z: calculated values [M+10H] ¹⁰⁺ 971.4, [M+11H] ¹¹⁺ 883.2; observed values [M+10H] ¹⁰⁺ 971.2, [M+11H] ¹¹⁺ 882.9.

Example 6

Synthesis of polypeptide compounds of SEQ ID NO:6

The synthesis method is the same as in Example 1. The target peak is collected and freeze-dried to obtain 0.013g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10284.4. ESI-MS m/z: calculated value [M+9H] ⁹⁺ 1143.7, [M+11H] ¹¹⁺ 935.9; observed value [M+9H] ⁹⁺ 1143.5, [M+11H] ¹¹⁺ 935.6.

Example 7

Synthesis of SEQ ID NO:7 polypeptide compounds

The synthesis method is the same as in Example 1. The target peak is collected and freeze-dried to obtain 0.010g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9329.3. ESI-MS m/z: calculated value [M+9H] ⁹⁺ 1037.6, [M+10H] ¹⁰⁺ 933.9; observed value [M+9H] ⁹⁺ 1038.9, [M+10H] ¹⁰⁺ 933.7.

Example 8

Synthesis of SEQ ID NO:8 polypeptide compounds

The synthesis method is the same as in Example 1. The target peak is collected and lyophilized to obtain 0.014g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9619.7. ESI-MS m/z: calculated value [M+9H] ⁹⁺ 1069.9, [M+10H] ¹⁰⁺ 963.0; observed value [M+9H] ⁹⁺ 1069.8, [M+10H] ¹⁰⁺ 962.8.

Example 9

Synthesis of polypeptide compounds of SEQ ID NO:9

The synthesis method is the same as in Example 1. The target peak is collected and lyophilized to obtain 0.015g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10200.3. ESI-MS m/z: calculated value [M+9H] ⁹⁺ 1134.4, [M+10H] ¹⁰⁺ 1021.0; observed value [M+9H] ⁹⁺ 1133.9, [M+10H] ¹⁰⁺ 1020.7.

Example 10

Determination of the agonistic activity of polypeptide compounds on human GLP-1 receptor, glucagon and Y ₂ receptor

To determine the agonistic effects of peptide compounds on receptors by functional assays, GLP-1 receptor and glucagon receptor agonistic activity was measured by measuring the cAMP response of HEK-293 cell lines stably expressing human GLP-1 receptor or glucagon receptor. Cells stably expressing GLP-1 receptor or glucagon receptor were divided into T175 culture flasks and grown overnight in culture medium (DMEM/10% FBS) to a nearly confluent state, then the culture medium was removed and replaced with calcium- and magnesium-free PBS. Cells were washed and then protease treated with Accutase enzyme. Wash the detached cells and resuspend them in assay buffer (20mM HEPES, 0.1% BSA, 2mM IBMX, 1×HBSS) and determine the cell density and aliquot 25 μL into 96-well plates. hole. For measurement, 25 μL of a solution of the test polypeptide compound in assay buffer is added to the wells and then incubated at room temperature for 30 min. Cisbio's kit was used to determine the cAMP content of cells based on homogeneous time-resolved fluorescence (HTRF). After adding HTRF reagent diluted in lysis buffer (kit component), the plate was incubated for 30 minutes and the fluorescence ratio at 665/620 nm was measured. The in vitro potency of agonists was quantified by detecting the concentration that elicited 50% activation of the maximal response ( _EC50 ).

HEK-293 cells stably expressing human _Y receptors and cAMP-sensitive calcium channels were used to determine the agonistic effects of compounds on _Y receptors. First, cells were cultured in culture medium (DMEM, 10% FBS, Geneticin, Geneticin, Penicillium/Streptomycin), and then 20 μL of cell suspension per well was added to a 384-well plate (20,000 cells /hole). Cells were then pretreated with calcium-sensitive dye for 50 min at 37°C, 5% _CO2 , followed by 10 min at 25°C. The compound to be tested was serially diluted 4 times 10 times, and 750 nL of the test compound was transferred to a 384-well plate. The 384-well plate is then removed from the incubator and placed into the FLIPR Tetra System. The fluorescence signal is measured (excitation 494nm/emission 516nm) by detecting the concentration that causes 50% activation of the maximum response ( _EC50 ). The in vitro potency of agonists was quantified.

The detection data (nM) in the examples of this patent application are shown in Table 1 below. Although the detection data are stated with a certain number of significant figures, it should not be considered that the data has been determined to be an exact number of significant figures.

Table 1: Agonistic activity of polypeptide compounds on human GLP-1 receptor, Glucagon receptor and Y ₂ receptor

As shown in Table 1, all polypeptide compounds showed triple agonist activity on GLP-1 receptor, glucagon receptor and Y ₂ receptor, indicating that these polypeptide compounds have the characteristics of triple agonists. At the same time, some peptide compounds show agonistic activities at GLP-1 receptors, glucagon receptors and Y ₂ receptors that are close to or better than GLP-1, glucagon and PYY _3-36 .

Example 11

Effects of peptide compounds on eating in ICR mice

Male ICR mice were randomly divided into 5 groups, with 6 mice in each group. Mice were fasted for 12 hours before the experiment. The blank group was given normal saline (10 mg/kg) by subcutaneous injection. The administration groups were divided into 4 groups. The mice were given a single subcutaneous injection of 30 nmol/kg of liraglutide and GEP44 (GLP- 1/Y ₂ receptor dual agonist, J. Med. Chem. 2021, 64, 2, 1127–1138), xGLP/GCG-15 (GLP-1/glucagon receptor dual agonist, European Journal of Medicinal Chemistry, 2021, 212, 113118) and SEQ ID NO: 6. Then the mice were immediately given pre-weighed rat food, and the weight of the food was weighed again at 2h, 4h, 6h, 12h and 24h, and the food intake of the mice at different time points was calculated.

As shown in the results in Figure 1, the results of the feeding experiment in ICR mice showed that at 24 hours, the SEQ ID NO:6 polypeptide compound significantly reduced the food intake of the mice, and its inhibitory effect on food intake was significantly better than liraglutide and GEP44 and xGLP/GCG-15, indicating that the polypeptide compound of the present invention has excellent eating inhibitory effect.

Example 12

Pharmacokinetic properties of peptide compounds in rats

SD rats were administered subcutaneous (s.c.) injection of 50 nmol/kg liraglutide and SEQ ID NO:6, and blood samples were collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 16h and 24h after administration. After protein precipitation using acetonitrile, plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-life were calculated using WinonLin 5.2.1 (non-compartmental model) (Table 2).

Table 2: Overview of pharmacokinetics of peptide compounds in rats

sample T _1/2 (h) C _max (ng/mL) Liraglutide 2.3 489 SEQ ID NO:6 4.6 398

As shown in the results in Table 2, the in vivo half-life of the polypeptide compound of the present invention is significantly extended, which is better than liraglutide, and has pharmacokinetic characteristics that support once-daily administration.

Example 13

Acute hypoglycemic activity of peptide compounds in mice

Male ICR mice were randomly divided into groups, with 6 mice in each group. Give only water and fast overnight. The blank group was given intraperitoneal injection of normal saline (10 mg/kg), and the administration groups were divided into 4 groups. The mice were given a single intraperitoneal injection of 30 nmol/kg of liraglutide and GEP 44 (GLP-1/Y ₂ receptor dual) in a non-fasting state. Agonist, J. Med. Chem. 2021, 64, 2, 1127–1138), xGLP/GCG-15 (GLP-1/glucagon receptor dual agonist, European Journal of Medicinal Chemistry, 2021, 212, 113118) and SEQ ID NO: 6. After 30 minutes, mice in each group were given 3g/kg glucose solution intraperitoneally. Use a blood glucose meter to measure blood sugar levels at -30min, 0min, 15min, 30min, 60min, and 120min

As shown in the results of Figure 2, the acute hypoglycemic experiment in ICR mice showed that the SEQ ID NO: 6 polypeptide compound significantly improved the glucose tolerance level of the mice and had excellent hypoglycemic effect, and its hypoglycemic effect was also excellent. For liraglutide and GEP 44.

Example 14

Effects of polypeptide compounds on blood lipids and body weight in diet-induced obesity (DIO) mice

Male C57BL/6J mice, weighing about 22g, and a total of 18 mice in the model group, were fed with D12492 high-fat feed from Research Diets Company for 18 weeks to create a DIO mouse model. Before the start of drug administration, DIO mice in each group were randomly divided into 5 groups according to their body weight, with 6 mice in each group, namely the normal saline group (blank control group) and the positive control group (liraglutide, GEP44, xGLP/GCG- 15) and the test sample group (SEQ ID NO: 6). Mice in each group were subcutaneously injected with normal saline (10mg/kg), liraglutide (30nmol/kg), xGLP/GCG-15 (30nmol/kg), SEQ ID NO: 6 (10nmol/kg) twice a day, and three times a day. GEP44 (30nmol/kg), administration cycle is 21 days. The body weight changes of mice were recorded every day. After the experiment, mice in each group were sacrificed, blood was taken to make serum, liver was taken to make homogenate, and the triglyceride (TG) and total cholesterol (TC) contents of liver and serum were measured respectively.

As shown in Figure 3, the polypeptide compound SEQ ID NO: 6 of the present invention can reduce the body weight of mice by 36.2% when administered continuously for 3 weeks in DIO mice at a dose of 10 nmol/kg. For the three positive control compounds, at a dose three times that of SEQ ID NO: 6 (30 nmol/kg), liraglutide could only reduce the body weight of mice by 15.1%, GEP44 could only reduce the body weight of mice by 24.9%, and xGLP/GCG- 15 could only reduce mouse body weight by 27.0%. The above results show that SEQ ID NO:6 can achieve significantly higher weight loss effects than liraglutide, GEP44 and xGLP/GCG-15 at low doses, indicating that it has excellent weight loss effects.

Table 3: Serum total cholesterol (TC) and triglyceride (TG) contents of DIO mice after 3 weeks of treatment

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the liraglutide, GEP44 and xGLP/GCG-15 groups (One-WayANOVA, Tukey post hoc test), the results are expressed as 6 for each group Mean ± SD per mouse.

Table 4: Liver total cholesterol (TC) and triglyceride (TG) contents of DIO mice after 3 weeks of treatment

Sample (dose) Total cholesterol (mg/g) Triglycerides (mg/g) Blank control (normal saline group) 14.26±1.59 94.28±6.71 Liraglutide(30nmol/kg) 12.11±1.25 ^* 73.07±4.78 ^*** GEP44(30nmol/kg) 11.65±0.86 ^** 77.72±6.62 ^*** xGLP/GCG-15(30nmol/kg) 10.15±0.75 ^*** 61.65±6.98 ^*** SEQ ID NO:6(10nmol/kg) 7.15±0.50 ^***,### 40.93±4.39 ^***,###

^* : P<0.05 compared with the blank control group; ^** : P<0.01 compared with the blank control group; ^*** : P<0.001 compared with the blank control group; ^### : Compared with liraglutide, GEP44 and xGLP/ GCG-15 group ratio P<0.001 (One-WayANOVA, Tukeypost hoc test), the results are expressed as the mean ± SD of 6 mice in each group.

As shown in Table 3 and Table 4, the polypeptide compound SEQ ID NO: 6 of the present invention can significantly reduce the triglycerides in the serum and liver of mice ( TG) and total cholesterol (TC) content, and the effect of SEQ ID NO:6 on reducing serum and liver blood lipids is significantly stronger than that of the positive control drugs liraglutide (30nmol/kg), GEP44 (30nmol/kg) and xGLP/ at high doses. GCG-15 (30nmol/kg). The above results show that SEQ ID NO: 6 at low doses can achieve weight loss and liver and serum blood lipid reduction effects that are significantly better than liraglutide, GEP44 and xGLP/GCG-15, indicating that the polypeptide compound of the present invention has exceptionally excellent weight loss effects. Weight loss, lipid regulation and treatment of NAFLD.

Claims (7)

1. A type of GLP-1/glucagon/Y ₂ receptor triple agonist, characterized in that the amino acid sequence of the GLP-1/glucagon/Y ₂ receptor triple agonist is one of the following sequences: (1)SEQ ID NO:1 (2)SEQ ID NO:2 (3)SEQ ID NO:3 (7)SEQ ID NO:7 (8)SEQ ID NO:8 (9)SEQ ID NO:9 . 2. A pharmaceutically acceptable salt of a GLP-1/glucagon/Y ₂ receptor triple agonist, characterized in that: the amino acid sequence of the GLP-1/glucagon/Y ₂ receptor triple agonist is as claimed in the claims One of the amino acid sequences described in 1. 3. A pharmaceutically acceptable salt of a type of GLP-1/glucagon/γ ₂ receptor triple agonist according to claim 2, wherein the salt is a GLP-1/glucagon/γ ₂ receptor triple agonist. Salts of agonists with one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid, succinic acid. 4. A medicament prepared from a GLP-1/glucagon/Y ₂ receptor triple agonist according to claim 1, characterized in that the medicament is any pharmaceutical tablet, capsule, Inhalants, sprays, injections, films, patches, emulsions, suppositories or compound preparations, the medicine consists of a type of GLP-1/glucagon/Y ₂ receptor triple agonist and pharmaceutically acceptable pharmaceutical excipients, carriers or Diluent composition. 5. A pharmaceutical composition containing a type of GLP-1/glucagon/Y ₂ receptor triple agonist, characterized in that the pharmaceutical composition is a GLP-1/glucagon/Y ₂ receptor triple agonist according to claim 1 The agent is the effective raw material, or the pharmaceutically acceptable salt of the GLP-1/glucagon/Y ₂ receptor triple agonist described in claim 2 or 3 is the effective raw material, plus a pharmaceutically acceptable carrier or diluent composition. 6. The GLP-1/glucagon/Y ₂ receptor triple agonist or its pharmaceutically acceptable salt, or its pharmaceutical composition or its medicament in any one of claims 1-5 is prepared for treatment Use in medicines for metabolic diseases or conditions. 7. Use according to claim 6, characterized in that the metabolic disease or condition is diabetes, obesity, non-alcoholic fatty liver disease and dyslipidemia.