WO2012155780A1

WO2012155780A1 - Branched-peg modified glp-1 analogue and pharmaceutically acceptable salts thereof

Info

Publication number: WO2012155780A1
Application number: PCT/CN2012/074706
Authority: WO
Inventors: 王瑞军; 赵军军
Original assignee: 江苏豪森药业股份有限公司; 江苏豪森医药集团连云港宏创医药有限公司
Priority date: 2011-05-19
Filing date: 2012-04-26
Publication date: 2012-11-22
Also published as: CN103492412B; CN102786590A; CN103492412A

Abstract

Provided are a branched-PEG modified GLP-1 analogue and pharmaceutically acceptable salts thereof, methods of making the same, a pharmaceutical composition comprising a therapeutically effective amount of the analogue, and applications thereof in the preparation of a medicine that treats and/or prevents diabetes mellitus type 2.

Description

Branched PEG-modified GLP-1 analogues and pharmaceutically acceptable salts thereof

Technical field

The present invention relates to a branched PEG-modified GLP-1 analogue, a pharmaceutically acceptable salt thereof, a process for the preparation thereof, and a pharmaceutical composition comprising a therapeutically effective amount of the analog, and a medicament for treating and/or preventing type II diabetes Aspect of the application. Background technique

In recent years, with the improvement of living standards, the modernization of living patterns and the aging of society, the prevalence of diabetes in the world is increasing year by year, especially those developing countries that are rich and poor. Diabetes has become the third serious chronic non-infectious disease following cancer, cardiovascular and cerebrovascular diseases, and is one of the leading causes of death and disability. In 1997, the WHO reported that there were 135 million people with diabetes in the world, and by the year 2000 it is estimated to reach 175 million. The latest survey report in China shows that the prevalence of diabetes in natural populations over 20 years old is 3.21%. It is estimated that there are at least 20 million diabetic patients in China, and more than 95% of them are type II diabetic patients. From the statistical analysis of relevant data in 1993, the cost of direct treatment for diabetes in the same year was as high as 2.216 billion yuan, and this cost does not include the cost of treatment for complications caused by diabetes, treatment and health expenditure outside the hospital, and indirect social economy. loss.

Current methods of controlling type 2 diabetes include diet, attention to exercise, and medication to regulate blood glucose levels. The most commonly used drugs include insulin, sulfonylureas, biguanides, and glitazones. These drugs emphasize that the body's blood sugar tends to be normal and cannot correct the complications caused by diabetes, especially the kidneys, cardiovascular system, vision and nervous system. These complications are directly related to the increase in mortality caused by diabetes. The main side effects of the first generation of drugs for the treatment of diabetes include hypoglycemia, weight gain, and edema. The mechanism of action of these drugs may vary, but there is no mechanism to protect the β-cells that secrete insulin, making it impossible to maintain normal blood glucose metabolism and endocrine regulation in the body. In many cases, the use of a single drug will slowly lose its effect, forcing a combination of treatments, often with lower blood pressure and cholesterol-lowering drugs, so the long-term effects of such a program are different. Therefore, research and development of new drugs to control blood sugar, in conjunction with the current drugs, emphasize the protection and repair of β-cell function, regulate the internal division The response of the secretion system to food intake will revolutionize the treatment of diabetes.

Research on the agonist of the glucagon-like peptide-1 (GLP-1) receptor is promising, and research and development in this field may open a new chapter in the field of treatment of type 2 diabetes. The glucagon-like peptide-1 was discovered in 1984 as an enteroendocrine hormone. If a hormone is administered to a type 2 diabetic, the blood glucose level can be adjusted to normal levels (Nathan, DM, et al. Diabetes Care 1992; 15:270-6; Zander, M, et al. Lancet 2002; 359:824- 30). Studies have shown that glucagon-like peptides and their receptor agonists are mainly responsible for the secretion of insulin by activating the glucagon analog-1 receptor on the surface of pancreatic β-cells. Because this effect is determined by the level of blood glucose in the body itself, it does not occur like traditional medicine, even in the presence of glucagon-like peptide-1 and its receptor agonist. Severe hypoglycemia causes life-threatening hypoglycemic shock. Specifically, when the blood glucose concentration in the body is higher than 6 mmol/L, the glucagon-like peptide-1 can have a remarkable effect of promoting insulin secretion, and when the blood glucose in the body tends to a normal level, it does not continue to function. In addition, such agonists also have the effect of stimulating rodent (rat:) islet β-cell growth and increasing beta-cell tissue. This ability to repair islet β-cells provides promise for the cure of type 2 diabetes, at least delaying the progression from type II to type I. Furthermore, glucagon-like peptide-1 and its receptor agonist simultaneously inhibit the secretion of glucagon and thereby reduce the possibility of hepatic blood glucose output. More significantly, such agonists are effective in inhibiting gastrointestinal motility and gastric emptying, resulting in reduced food intake and reduced body weight. This can help control the weight of people with type 2 diabetes.

CN1976948A discloses a long-acting polyethylene glycol (PEG)-modified insulin secretagogue peptide compound and a pharmaceutically acceptable salt thereof, which have the function of activating glucagon-like peptide-1 (GLP-1) receptor Insulin secretion, lowering blood sugar, so it can effectively treat or prevent type 2 diabetes. This patent application gives a sequence of a series of GLP-1 receptor agonists, which are also indicated by the modification of the sequence by polyethylene glycol. Currently, PEG for modification has various structures, such as two-branched type, four-branched type, etc., and branched type PEG has various kinds of linkers, such as a glycerin linker, a lysine linker and the like. For each specific structure of the biopeptide, which PEG modification method is optimal is not fixed. After studying various ways, the inventors unexpectedly discovered the specific order for the present invention. In terms of columns, a particular modification produces surprisingly good results. Summary of the invention

It is an object of the present invention to provide a branched PEG-modified GLP-1 analog of the formula ω and a pharmaceutically acceptable salt thereof:

HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC-NH ₂

(I)

Wherein the branched PEG is selected from the group consisting of two-branched or four-branched; the molecular weight of the branched PEG is 20-60 KD; and the linker of the branched PEG is selected from glycerol or lysine.

From the experimental example 1 of the present invention, each test substance agonizes the GLP-1 receptor EC ₅ . (mM) It can be seen that as the molecular weight of PEG increases (from 20KD for PEX-167 to 40KD for PEX-168) and the number of PEG branches increases (from the straight chain of PEX-165 to the two branches of PEX-167, To the four branches of PEX-166:), the in vitro activity of the modified polypeptide was significantly reduced. Although changes in in vivo activity with changes in PEG molecular weight and PEG branching status are not necessarily consistent with changes in in vitro activity, maintenance of in vitro activity is a necessary condition for excellent in vivo activity. Meanwhile, regarding the in vivo activity (hypoglycemic effect) of the compound provided by the present invention, it can be seen from Table 2 of Experimental Example 2 of the present invention that the long-acting effect of the linear PEG modification is weaker than the modification of the branched PEG. The effect, while the two-branch PEG modification, the larger the molecular weight, the longer the activity in vivo, that is, the long-term effect produced by the modification of the large molecular weight and branched PEG is obvious. In summary, the two-branched PEG has a prominent advantage in modifying the peptide chain of the present invention. Therefore, the branched PEG of the present invention is preferably of a two-branched type; the molecular weight of the branched PEG is preferably 40 KD or 60 KD.

As can be seen from Table 3 of Experimental Example 3, the inhibitory effect of PEX-168 on body weight was weaker than that of PEX-170, indicating that the modification effect of PEG on the lysine (Lys) linker is better than that on the glycerol linker. Therefore, the linker of the branched PEG of the present invention is preferably lysine (Lys) o

More preferably, the PEG is a branched type mPEG2-Lys-MAL (40kD) o More preferably, the structure of the branched PEG-modified GLP-1 analogue of formula (II) His-D-Ala - Glu-Gly - Thr-Phe-Thr- Ser-Asp-Leu-Ser- Lys-Gin-Nle-Glu-Glu-Glu-Ala-Val-Arg- Leu - Phe - lie - Glu - Trp - Leu - Lys - Gin - Gly - Gly - Pro - Ser - Ser - Gly - Ala - Pro

Another object of the present invention is to provide a method for preparing the branched PEG-modified GLP-1 analogue and a pharmaceutically acceptable salt thereof, which comprises a method of synthesis, purification and drying, which is preferably a solid phase or a liquid phase. Synthesis, the purification method is preferably a reverse phase high performance liquid phase, ion exchange or gel filtration purification method, and the drying is preferably freeze drying.

Another object of the present invention is to provide a use of the branched PEG-modified GLP-1 analog and a pharmaceutically acceptable salt thereof for the treatment and/or prevention of type 2 diabetes.

Based on CN1976948A, the present invention optimizes the design and extensive screening of PEG for modification, and obtains a longer-lasting and more active GLP-1 analog, and the compound provided by the invention has simple preparation process and low cost. It is more suitable for industrial production. DRAWINGS

Figure 1: HPLC chromatogram and analysis conditions of SEQ ID N0.95.

Figure 2: ESI/MS mass spectrum of SEQ ID N0.95.

Figure 3: Maldi-TOF map of PEX-168.

Figure 4: Effect of single administration on daily fasting blood glucose in db/db mice (n=10) _o Specific embodiment

In order to more specifically describe the present invention, the following specific embodiments are provided, but the scope of the present invention is not limited thereto. The basic situation of the specific compounds involved in this section can be seen in the specific situation. List of compounds.

Specific compound list

Example 1: Synthesis of GLP-1 Analogue SEQ ID N0.95

The sequence of SEQ ID N0.95 is shown in formula (I): HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC-NH ₂

(I)

According to the structural characteristics, it can be synthesized by the already mature solid phase synthesis technology. 1.1 Amino acid derivatives used in the synthesis

Fmoc-His(Trt)-OH, Fmoc-D-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ser (tBu)-OH, Fmoc-Asp(OtBu)-OH , Fmoc-Leu-OH , Fmoc-Lys(Boc)-OH , Fmoc-Gln(Trt)-OH , Fmoc-Nle-OH , Fmoc-Ala-OH , Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH.

The above amino acid derivatives were purchased from Jill Biochemistry.

1.2 Resins and other reagents

Resin: Rink Amide-AM resin (; Gil Biochemical).

Other reagents: Ν, Ν'-diisopropylcarbodiimide (DIC), hydroxybenzotriazole (ΗΟΒΤ), dimethylformamide (DMF), dichloromethane (DCM), trifluoroacetic acid (TFA), Tis, piperidine (PIP), acrylonitrile (ACN).

1.3 Synthesis process

Rink Amide-AM resin (0.7mmol/g) 20g as solid phase carrier, Fmoc-AA-OH (5 times excess, 70mmol) as raw material, DIC (5 times excess, 70mmol), HOBT (5-fold excess, 70 mmol) is a condensing agent, according to the sequence of SEQ ID N0.95

The C-terminal is sequentially synthesized to the N-terminus, the amino acid condensation time is about 2-4 hours per step, the Fmoc protecting group removal reagent is 20% piperidine/DMF, and the condensation and de-Fmoc endpoint detection is performed by the ketone test method (Kaiser Test). .

After the condensation is completed, a resin peptide is obtained. After drying, the resin peptide was cleaved with TFA/Ti _S /H ₂ 0 (95 _: 2.5 _: 2.5:) as a cleavage reagent, reacted at room temperature for about 3 hours, the resin was filtered off, and the lysate was passed to B.

52.0g. The obtained crude product was dissolved in purified water , = EtOAc (m.).

The molecular weight of the obtained compound was determined by ESI-MS (yield: 4211.7).

Example 2: Synthesis of Maleimide-Based Reactive Polyethylene Glycol

This example is a synthesis of a linear, branched maleimide-based reactive polyethylene glycol, wherein the main raw materials are mPEG-OH (5kD), mPEG-OH (10kD), mPEG-OH (20kD), mPEG. -OH (30kD) are purchased from Sunbio, Korea, and other reagents such as ethylenediamine and N-hydroxysuccinimide (HOSi, Lys, toluene, and dichloromethane) are common reagents.

Synthesis of 2.1 mPEG-MAL (20kD)

Triphosgene 〇

OH

,σ 〇〇- -CI mPEG-OH(20KD)

〇

〇 NH

H. N

HOSu

TEA

SC-m PEG (20kD)

〇

mPEG-NHCH2CH2NH2 (20KD)

mPEG-MAL (20KD) 20 g (lmmol) of mPEG-OH (20 kD) was placed in a 200 ml single-mouth bottle, 100 ml of toluene was added, and the water was refluxed; then toluene was distilled off, cooled to room temperature, and then 100 ml of DCM was added, followed by the addition of 1.18 g (4 mmol) of tri-light. The mixture was stirred overnight at room temperature; the reaction solution was poured into 200 ml of anhydrous diethyl ether in a ventilated kitchen, filtered, and dried in vacuo to give a white solid 15 g. 15 g of this white solid was placed in a 200 ml single-mouth bottle, 100 ml of a toluene/DCM (2:1) solution was added, 0.25 g of HOSu was added, followed by 0.3 g of triethylamine, and the reaction was stirred at room temperature overnight; after the reaction was completed The reaction solution was filtered, and the filtrate was directly poured into 100 ml of anhydrous diethyl ether, filtered, and dried in vacuo to give a white solid, 14 g, which is SC-m PEG (20 kD) _o

1.4 g of anhydrous ethylenediamine was dissolved in a 200 ml reaction flask with 50 ml of DCM, and 14 g of SC-mPEG (20 kD) was dissolved in 100 ml of DCM and dissolved in the above ethylenediamine solution, and allowed to react overnight; the reaction was stopped the next day. Filtration, the filtrate was added to 500 ml of saturated brine, and the aqueous layer was extracted with EtOAc EtOAc EtOAc. and dried in vacuo to give a white solid 13g, that is, _{_{_{mPEG-NHCH 2 CH 2 NH 2}}} (20kD).

13 g of mPEG-NHCH ₂ CH ₂ NH ₂ (20 kD) and 0.8 g of MAL-ONP were dissolved in 100 ml of DCM, and 0.3 g of triethylamine was added thereto, and the reaction was stirred at room temperature overnight; then the reaction mixture was filtered, and the filtrate was concentrated to dryness. Then, 100 ml of ethyl acetate was added and dissolved by heating, and the solid was precipitated, filtered, and dried in vacuo to give 12 g of white solid, m.

The molecular weight of the product measured by MALDI-TOF-MS was 20522.5 (theoretical value: 20000 ± 2000).

Synthesis of 2.2 mPEG4-Lys-MAL (20kD)

1.73 g (5 mmol) of BOC-Lys(BOC)-OH was dissolved in 200 ml of a mixed solution of DCM and DMF, and cooled to about 0 ° C in an ice bath. After adding 0.69 g (6 mmol) of HOSu, 0.76 g (0.76 g) was added dropwise. 6 mmol) DIC of DCM solution, after completion of the dropwise addition, the reaction was maintained at 0 ° C for 6 h, then the ice bath was removed, and the reaction was allowed to stand overnight at room temperature; then the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure, crystallised in anhydrous diethyl ether and hexanes and filtered. Vacuum drying gave 2 g of a white solid, i.e., BOC-Lys(BOC)-OSu (intermediate 1), yield 87%.

After 270 mg (1.5 mmol) of lysine hydrochloride (Lysine'HCL) was adjusted to a pH of 8.0 with a 1 mol/L NaOH solution, 1.84 g (4 mmol) of DCC of BOC-Lys(BOC)-OSu was added. The mixed solution with DMF was added to the above solution to maintain the pH at 8.0, and after adjusting for 4 hours, the pH was adjusted to 3 with oxalic acid, using saturated brine. Washing to neutrality, and then extracting with DCM, EtOAc EtOAc (EtOAc m. 83%.

After lg intermediate 2 was dissolved in 20 ml of DCM, 5 ml of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 30 minutes, and then concentrated to dryness to give intermediate 3.

After dissolving 360 mg (0.9 mmol) of intermediate 3 in 100 ml of 0.2 M borate buffer to pH 8.0, 20 g (4 mmol) of SC-mPEG (5 kD) (Synthesis of SC-mPEG (5 kD), reference 2.1) was added. After maintaining the pH at 8.0, the reaction was stirred overnight; after the reaction was completed, it was diluted with 600 ml of purified water and adjusted to pH 3 with oxalic acid, and extracted with DCM. The precipitated solid was precipitated, collected by filtration, and dried in vacuo to give a white solid (17.6 g, which was crude product of intermediate 4, and the crude product was purified by strong basic anion exchanger QAE-Sephadex A-50, removing 5 kD, 10 kD, 15 kD components, and combining 20 kD. component, extracted with DCM, the organic layers were combined, dried over anhydrous Na ₂ S0 _4, filtered, and the filtrate was concentrated under reduced pressure, the precipitated solid was settling in dry ether, filtered and dried in vacuo to give a white solid 12g, that is, pure intermediate 4 .

8 g (0.4 mmol) of the intermediate 4 was dissolved in 150 ml of DCM, and 0.6 g of HOSu and 0.65 g of DIC were added and allowed to react at room temperature overnight; then the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. Filtration and drying in vacuo gave 7.5 g of a white solid, m.p.

7.5 g (0.38 mmol) of mPEG4-Lys-NHS (20 kD) was dissolved in 80 ml of DCM, and then 0.9 g of anhydrous ethylenediamine dissolved in 40 ml of DCM was added dropwise to the above solution with a dropping funnel, and added dropwise. After the completion of the reaction, the reaction mixture was filtered overnight. The filtrate was filtered. The filtrate was filtered and evaporated, evaporated, evaporated, evaporated, evaporated. the precipitated solid was filtered and dried in vacuo to give a white solid 7g, namely _{mPEG4-Lys-NH (CH 2} ) 2 NH 2 (20kD), 93% yield.

7 g (OJmmol) mPEG4-Lys-NH(CH ₂ ) ₂ NH ₂ (20 kD) and 0.4 g of MAL-ONP were dissolved in 100 ml of DCM, 0.15 g of triethylamine was further added, and the reaction was stirred at room temperature overnight; then the reaction solution was filtered. The filtrate was concentrated to dryness under reduced pressure, and then dissolved in ethyl acetate (100 ml), and the solid was dissolved, filtered, and dried in vacuo to give 6 g of white solid. It was mPEG4-Lys-MAL (20 kD), and the yield was 86%.

The product molecule was measured by MALDI-TOF-MS: 21021.7 (theoretical value:

20000±2000).

Synthesis of 2.3 mPEG2-Lys-MAL (20kD)

The synthesis of SC-mPEG (10kD) is referred to the synthesis step of SC-mPEG (20kD) in 2.1.

After 90 mg (0.5 mmol) of lysine hydrochloride (Lysine'HCL) was adjusted to pH 8.0 with 100 ml of 0.2 M boric acid buffer, 12 g (1.2 mmol) of mPEG-OSu (lOkD) was added to maintain the pH at 8.0, Stirring reaction overnight; After the end of the reaction, the pH was adjusted to 3 with oxalic acid, then added with sodium chloride to sat. EtOAc (EtOAc). The precipitated solid was precipitated, collected by filtration, and dried in vacuo to give a white solid (11.5 g) as crude m.p. 20kD components were combined, extracted with DCM, the organic layers were combined, dried over anhydrous Na ₂ S0 _4, filtered, and the filtrate was concentrated under reduced pressure, the precipitated solid was settling in dry ether, filtered and dried in vacuo to give 9.3 g of a white solid, that is mPEG2- Lys-OH (20kD) pure product, yield 93%.

8 g (0.4 mmol) of mPEG2-Lys-OH (20 kD) was dissolved in 150 ml of DCM, 0.6 g of HOSu and 0.65 g of DIC were added and allowed to react at room temperature overnight; then the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The solid was precipitated by diethyl ether, filtered, and dried in vacuo to give 7.7 g of white solid, m.p.

7.7 g (0.38 mmol) of mPEG4-Lys-NHS (20 kD) was dissolved in 80 ml of DCM, and then 0.9 g of anhydrous ethylenediamine dissolved in 40 ml of DCM was added dropwise to the above solution with a dropping funnel, and added dropwise. The reaction was closed overnight; the reaction solution was filtered the next day, and filtered. The mixture was washed with aq. EtOAc (3 mL). That is, mPEG2-Lys-NH(CH ₂ ) ₂ NH ₂ (20 kD), yield 93%.

7.0 g (0.7 mmol) of mPEG2-Lys-NH(CH ₂ ) ₂ NH ₂ (20 kD) and 0.4 g of MAL-ONP were dissolved in 100 ml of DCM, 0.15 g of triethylamine was further added, and the reaction was stirred at room temperature overnight; The filtrate was filtered, and the filtrate was evaporated to dryness.

The molecular weight of the product measured by MALDI-TOF-MS was 20815.2 (theoretical value: 20000 ± 2000).

The synthesis of SC-mPEG (20 kD) is referred to the synthesis step of SC-mPEG (20 kD) in 2.1.

After dissolving 60 mg of lysine hydrochloride (Lysine'HCl) in 160 ml of 0.1 M boric acid buffer, pH 8.0, 21.6 g of SC-mPEG (20 kD) was added to maintain the pH at 8.0, and the reaction was stirred at room temperature for 24 hours. ; after the reaction, purification was diluted with 600ml water and adjusted to pH 3 with oxalic acid and extracted with DCM, and the organic layers were combined, dried over anhydrous Na ₂ S0 _4, filtered, and the filtrate was concentrated under reduced pressure, the precipitated solid was settling in anhydrous diethyl ether, was collected by filtration , dried in vacuo to give a white solid 19 g, which is mPEG2-Lys-OH (40 kD) crude; the crude product is purified with a strong basic anion exchanger QAE-Sephadex A-50, the 20 kD component is removed, the 40 kD component is combined, and extracted with DCM. The organic layers were combined, dried over anhydrous Na ₂ S0 _4, filtered, and the filtrate was concentrated under reduced pressure, the precipitated solid was settling in dry ether, filtered and dried in vacuo to give a white solid LLG, namely mPEG2-Lys-COOH (40kD) pure product.

10 g of mPEG2-Lys-OH (40 kD) was dissolved in 200 ml of DCM, and 0.3 g of HOSu and 0.3 g of DIC were added, and the reaction was carried out at room temperature overnight; then the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. It was vacuum dried to give 10 g of a white solid, m.p.

10 g of mPEG2-Lys-NHS (40 kD) was dissolved in 100 ml of DCM, and then 0.6 g of anhydrous ethylenediamine was added dropwise to the solution dissolved in 50 ml of DCM with a dropping funnel while stirring, and the reaction was sealed overnight; The reaction mixture was filtered, and the filtrate was washed with EtOAc EtOAc EtOAc EtOAc. 10 g of white solid, i.e., mPEG2-NH(CH ₂ ) ₂ NH ₂ (40 kD).

10 g of mPEG2-NH(CH ₂ ) ₂ NH ₂ (40 kD) and 0.3 g of MAL-ONP were dissolved in 100 ml of DCM, and then O.lg of triethylamine was added thereto, and the reaction was stirred at room temperature overnight; then the reaction solution was filtered, and the filtrate was evaporated. The mixture was concentrated to dryness. EtOAc (EtOAc m.

The molecular weight of the product measured by MALDI-TOF-MS was 41135.8 (theoretical value: 40000±4000)

Synthesis of 2.5 mPEG2-Lys-MAL (60kD) The synthesis of SC-mPEG (30 kD) is referred to the synthesis step of SC-mPEG (20 kD) in 2.1.

After dissolving 40 mg of lysine hydrochloride (Lysine'HCl) in 160 ml of 0.1 M boric acid buffer, pH 8.0, 21.6 g of SC-mPEG (30 kD) was added to maintain the pH at 8.0, and the reaction was stirred at room temperature for 24 hours. ; after the reaction, purification was diluted with 600ml water and adjusted to pH 3 with oxalic acid and extracted with DCM, and the organic layers were combined, dried over anhydrous Na ₂ S0 _4, filtered, and the filtrate was concentrated under reduced pressure, the precipitated solid was settling in anhydrous diethyl ether, was collected by filtration , dried under vacuum to obtain 19 g of a white solid, which is a crude product of mPEG2-Lys-OH (60 kD); the crude product is purified by a strong basic anion exchanger QAE-Sephadex A-50, the 30 kD component is removed, and the 60 kD component is combined and extracted with DCM. The organic layers were combined, dried over anhydrous Na ₂ S0 _4, filtered, and the filtrate was concentrated under reduced pressure, the precipitated solid was settling in dry ether, filtered and dried in vacuo to give a white solid 10g, that is, mPEG2-Lys-OH (60kD) pure product.

10 g of mPEG2-Lys-COOH (60 kD) was dissolved in 200 ml of DCM, 0.2 g of HOSu 0.2 g DIC was added, and the reaction was carried out at room temperature overnight; then the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. Vacuum drying gave 10 g of a white solid, m.p.

10 g of mPEG2-Lys-NHS (60 kD) was dissolved in 100 ml of DCM, and then 0.4 g of anhydrous ethylenediamine was added dropwise to the solution dissolved in 50 ml of DCM with a dropping funnel, and the reaction was allowed to complete overnight after the dropwise addition; The reaction mixture was filtered, and the filtrate was washed with EtOAc EtOAc EtOAc EtOAc. 10 g of white solid, i.e., mPEG2-NH(CH ₂ ) ₂ NH ₂ (60 kD).

10 g of mPEG2-NH(CH ₂ ) ₂ NH ₂ (60 kD) and 0.2 g of MAL-ONP were dissolved in 100 ml of DCM, and then 0.07 g of triethylamine was added thereto, and the reaction was stirred at room temperature overnight; then the reaction solution was filtered, and the filtrate was concentrated. To dryness, add 100 ml of ethyl acetate to dissolve and dissolve. Place the precipitated solid, filter, and dry in vacuo to obtain 9 g of white solid, mPEG2-Lys-MAL (60 kD) _o

The molecular weight of the product measured by MALDI-TOF-MS was 62604.8 (theoretical: 60000 ± 6000).

Example 3: Synthesis of PEGylated GLP-1 Analog SEQ ID N0.95 Reactive polyethylene glycol mPEG-MAL (20kD), mPEG4-Lys-MAL (20kD), mPEG2-Lys-MAL (20kD), mPEG2-Lys-MAL (40kD), mPEG2-Lys prepared in Example 2, respectively -MAL (60kD) and mPEG2-glycerol-MAL (40kD) (No. 2D3Y0T01) purchased from NEKTAR, USA, reacted with GLP-1 analogue SEQ ID N0.95 to form a thioether bond by Michael addition reaction. The polypeptide is covalently bound to polyethylene glycol to give the PEGylated GLP-1 analog SEQ ID N0.95.

Structure of mPEG2-glycerol-MAL (40kD)

3.1 Preparation of PEX-165

Weigh 5.0 g of mPEG-MAL (20 kD) and 1.26 g of GLP-1 analogue SEQ ID N0.95 (1.2 fold excess), add 300 ml of 0.1 M sodium phosphate buffer (pH 7.7), and stir the reaction for 2 hours at room temperature. .

The reaction solution was separated and purified using a reverse phase HPLC preparative column (Lima, C18), and lyophilized to give a white solid (2.5 g) as PEX-165.

The molecular weight of PEX-165 measured by MALDI-TOF-MS was 24663.5 (theoretical value: 24211±2000).

3.2 Preparation of PEX-166

Weigh 5.0 g of mPEG4-Lys-MAL (20 kD) and 1.26 g of GLP-1 analogue SEQ ID N0.95 (1.2 fold excess), add 300 ml of 0.1 M sodium phosphate buffer (pH 7.7), and stir the reaction at room temperature. 2 hours.

The reaction solution was separated and purified using a reverse phase HPLC preparative column (Lima, C18), and lyophilized to give a white solid (2.5 g) as PEX-166.

The molecular weight of PEX-166 measured by MALDI-TOF-MS was 25640.8 (theoretical value: 24211±2000).

3.3 Preparation of PEX-167

Weigh 5.0 g of mPEG2-Lys-MAL (20 kD) and 1.26 g of GLP-1 analogue SEQ ID N0.95 (1.2 times excess), 300 ml of 0.1 M sodium phosphate buffer (pH 7.7) was added, and the reaction was stirred at room temperature for 2 hours.

The reaction solution was separated and purified using a reversed phase HPLC preparative column (Lima, C18), and lyophilized to give a white solid (2.2 g) as PEX-167.

The molecular weight of PEX-167 measured by MALDI-TOF-MS was 24988.0 (theoretical value: 24211±2000).

3.4 Preparation of PEX-168

Weigh lO.Og mPEG2-Lys-MAL (40kD) and 1.26g of GLP-1 analogue SEQ ID N0.95 (1.2 times excess), add 300ml 0.1M sodium phosphate buffer (pH 7.7), stir at room temperature Reaction for 2 hours.

The reaction solution was separated and purified using a reverse phase HPLC preparative column (Lima, C18), and lyophilized to give a white solid 4.5 g, which is PEX-168.

The molecular weight of PEX-168 measured by MALDI-TOF-MS was 44884.4 (theoretical value: 44211±4000).

3.5 Preparation of PEX-169

Weigh 15.0 g of mPEG2-Lys-MAL (60 kD) and 1.26 g of GLP-1 analogue SEQ ID N0.95 (1.2 fold excess), add 300 ml of 0.1 M sodium phosphate buffer (pH 7.7), and stir the reaction at room temperature. 2 hours.

The reaction solution was separated and purified using a reverse phase HPLC preparative column (Lima, C18), and lyophilized to give a white solid 6.2 g, which is PEX-169.

The molecular weight of PEX-169 measured by MALDI-TOF-MS was 67630.6 (theoretical value: 64211 ± 6000).

3.6 Preparation of PEX-170

Weigh 10.0 g of mPEG2-glycerol-MAL (40 kD) and 1.26 g of GLP-1 analogue SEQ ID N0.95 (1.2 fold excess), add 300 ml of 0.1 M sodium phosphate buffer (pH 7.7), and stir the reaction at room temperature. 2 hours.

The reaction solution was separated and purified using a reverse phase HPLC preparative column (Lima, C18), and lyophilized to give a white solid 4.5 g, which is PEX-170.

The molecular weight of PEX-170 measured by MALDI-TOF-MS was 4,4506.9 (theoretical: 44211 ± 4000). Experimental Example 1: Glucagon-like peptide-1 receptor agonistic activity assay

1. Test drug and positive control drug

1.1 Tested drugs: PEX-165, PEX-166, PEX-167, PEX-168, PEX-170. Storage method: Keep away from light and store at -20 °C.

Preparation method: Weigh a certain amount of the above compound, dilute to 100 μ ₈ / ηι1 mother liquor with dimethyl sulfoxide (DMSO), and then use 10-fold gradient dilution with DMSO, the final concentration is 1000 ng / ml, 100 ng /mK 10 ng/mK 1 ng/mK 10 ^g/mK 10 ² ng/ml and 10 ng/ml.

Dosage group: All test drugs are set at 7 concentrations, and each concentration is set to 3 duplicate wells. 1.2 Positive control drug: SEQ ID N0.95 o

Storage method: Keep away from light and store at -20 °C.

Method of preparation: A certain amount of SEQ ID N0.95 was weighed and the compound was diluted to a concentration of 10 nM positive control drug with dimethyl sulfoxide (DMSO).

2. Reagents and instruments

2.1 main reagent

DMEM medium (GIBCO, Cat No 12800017)

Dimethyl sulfoxide (Genebase, Prod No: 0231)

Luciferase Activity Assay Kit (Promega, Prod No: E2550)

2.2 Main instruments

Envision 2101 Multi-Purpose Microplate Reader (PerkinElmer)

3. Experimental principles and methods

3.1 Experimental principle

Glucagon-like Peptide-1 (GLP-1) produced by the large intestine is highly specific through the GLP-1 receptor (GLP-1 Receptor, GLP-1 R) of islet β cells. The combination of adenylate cyclase activates cAMP and further activates protein kinase. Metabolic signaling (glycobolism) and kinase signaling (GLP-1 binding) act synergistically at the cell membrane level, ultimately leading to Ca ²⁺ channel opening, Ca ²⁺ influx, thereby stimulating insulin secretion, while inhibiting glucagon production, Postprandial blood glucose is lowered to maintain a constant level.

Based on the GLP-1R signal transduction pathway, a HEK293 cell line stably expressing GLP-1R and a cAMP-driven luciferase reporter gene was constructed for screening of GLP-1R agonists. Selected. When GLP-1R binds to the agonist, the intracellular cAMP concentration increases and the expression of the cAMP-driven luciferase reporter gene is up-regulated. The ability of a compound to agonize GLP-1R activity can be determined by detecting luciferase activity.

3.2 Experimental steps

(i) HEK293 cells stably expressing GLP-1R and luciferase reporter gene were seeded in a 96-well culture plate at a cell volume of 50,000 cells/well in 10% FBS high glucose DMEM at 37 °C. Incubate for 24 hours under 5% C0 ₂ conditions.

(ii) The compound to be sieved was diluted with DMSO at the required concentration, and then added to the above 96-well microplate at 1 μM/well, and shaken gently with shaking. The 1 and 12 columns of the culture plate were used as positive controls. Incubate for 5 hours at 37 ° C under 5% CO ₂ .

(iii) Aspirate 50 μM of medium per well, add 50 μL of luciferase activity assay reagent, and shake for 10 minutes.

(iv) Aspirate 80 μΐ of the above mixture into each well, transfer to a 96-well plate, and measure the chemiluminescence count on an Envision 2101 Multi-Purpose Plate Microplate Reader.

(v) Data processing.

4. Data processing and statistical analysis

Using SEQ ID N 0.95 as a positive compound, the activation rate (%Response) at each concentration of each sample was calculated by the following formula.

VoResponse = L Sample - LBlank _{x 1 00%}

LSEQ NO.95- LBlank a _mpfe indicates the detection signal value after sample stimulation, L _M indicates blank, that is, the detection signal value of DMSO well, LSEQ NO. indicates 10 nM positive control sample SEQ ID N0.95 After detection signal value after stimulation .

The EC _5Q value is calculated by the non-linear fitting of the sample concentration of ^R^/^w by the following formula, Γ is the response high value, and SWto is the response low value.

Top-Bottom

VoResponse = Bottom +

1+10 (LogECso-X)

5. Test results

The results are shown in Table 1. Table 1 GLP-1 receptor EC standard error (nM) for each test substance 95% confidence limit EC ₅₀ (ng/ml) EC _5Q (nM)

(nM)

PEX165 1.748 0.087 0.007 0.072 to 0.102

PEX166 7.937 0.397 0.019 0.356 to 0.438

PEX167 2.507 0.125 0.010 0.104 to 0.146

PEX168 8.291 0.207 0.020 0.164 to 0.250

PEX170 34.675 0.254 0.013 0.226 to 0.282 Conclusion: The above compounds have GLP-1 receptor agonistic activity in vitro, wherein the order of activity from strong to weak is PEX-165, PEX-167, PEX-168, PEX-170, PEX - 166 , which indicates that the two-branched PEG of the Lys linker is more suitable for the specific sequence of the invention SEQ ID N 0.95.

Experimental Example 2: Effect of single administration on daily fasting blood glucose in type 2 diabetes db/db mice

1. Test drugs: PEX-165, PEX-166, PEX-167, PEX-168, PEX- 169ο Storage method: Protected from light, sealed at -20 °C.

Preparation method: Weigh different amounts of the above compounds, completely dissolve and dilute with PEX special solvent, and prepare 20 (^g/ml PEX-165, PEX-166, PEX-167 colorless transparent solution, 40 (^g /ml of PEX-168 colorless transparent solution and 60 (^g/ml PEX-169 colorless transparent solution (the above molar concentrations are equal:).

Dosage group: blank control group: PEX special solvent; PEX-165, PEX-166, PEX-167 group (200 g/ml); PEX-168 group (400 g/ml); PEX-169 group (60 (^ g/ml).

Route and volume of administration: A single subcutaneous injection is administered at a dose of 10 ml/kg.

2. Reagents and instruments

2.1 main reagent

PEX-168 special solvent: Jiangsu Haosen Pharmaceutical Co., Ltd., batch number: 20100719.

Sodium Chloride Injection: Shanghai Huayuan Changfu Pharmaceutical Co., Ltd., batch number: 10060201. 2.2 Main instruments

ΤΜ TM

Johnson &Johnson's basic plus blood glucose monitor ONE TOUCH BASIC Plus. 3. Test method

3.1 Type 11 diabetes (3⁄4/(3⁄4 mice screening, grouping and dosing)

Eighty db/db mice (40 males and 40 females) were purchased from animal houses at 4-5 weeks of age, fed in single cages, fed with high fat diet, and started at 7-8 weeks of age. Mice were fasted at 8:30 am 1 day before dosing (no water:), and fasting blood glucose was measured 6 hours later. Sixty db/db mice were selected, and their fasting blood glucose was between 10.2-24.7mmol/L. According to the fasting blood glucose of mice, the 60 mice were divided into 6 groups, 10 in each group (5 male and 5 female:) , were blank control group and 5 PEX compound administration groups.

From the day after the administration to 5 days after the administration (120 hours:), each group of mice was fasted at 8:30 am every day, and fasting blood glucose was measured 6 hours later.

3.2 Observation indicators

Fasting blood glucose: The fasting blood glucose of each group of mice was measured periodically.

4. Data processing and statistical analysis

Data were expressed as mean ± standard deviation (±s), and statistical analysis was performed on the data.

5. Test results

The results are shown in Figure 4 and Table 2.

On the 2nd day after db/db mice were injected subcutaneously with different PEX subjects, the fasting blood glucose of PEX-165 mice was not significantly different from the blank control group (P>0.05). The fasting blood glucose of the other groups was significantly lower than that of the blank control group. The control group (P<0.01, P<0.001) o On the third day after administration, the fasting blood glucose of PEX-166 and PEX-167 mice was not significantly different from the blank control group (P>0.05). On the 4th day after administration, the fasting blood glucose of the PEX-169 group was significantly lower than that of the blank control group (P<0.05). On the 5th day after administration, there was no significant difference in fasting blood glucose between the PEX-administered mice and the blank control group (P>0.05).

Therefore, a single subcutaneous injection of PEX compound can significantly reduce fasting blood glucose in db/db mice, and the duration of this effect is related to the structure of the compound. PEX-165 can reduce the duration of fasting blood glucose after a single subcutaneous injection until 1-2 days after administration. PEX-166 and PEX-167 can last 2-3 days after administration. PEX-168 can be administered continuously. After 3-4 days, PEX-169's hypoglycemic effect can be maintained for 4-5 days after administration. Table 2 Effect of single administration on daily fasting blood glucose in db/db mice

Soil s n=10) days after administration

Group before administration -

1 2 3 4 5 blank pair

17·91±3·46 17.50±3.86 17.98±3.28 18.49±3.53 18.60±2.45 18.26±2.51 Zhao

PEX-165 17.30±3.48 12.71±2.31 16.06±3.33 17.61±2.66 18.26±2.10 18.69±2.23

PEX-166 17.84±2.20 10.66±2.42 13.07±2.23" 16.94±2.79 17.32±2.65 17.26±3.07

PEX-167 17.44±3.17 9.01±2.80 11.22±2.55 15.45±2.92 18.16±3.03 17.9±2.16

PEX-168 17.73±2.98 9.12±3.13 11.04±2.60 13.38±2.27** 16.05±3.15 16.68±2.12

PEX-169 17.49±3.99 6.39±2.60 8.13±2.49 10.56±2.01 13.99±3.87* 16.22±2.29

*Ρ<0.05, **Ρ<0.01, ***Ρ<0.001 compared with the blank control group

Conclusion: After a single dose of the above five test substances (equimolar concentration:), PEX-166 is longer than PEX-165, PEX-168 is longer than PEX-167, and PEX-169 is longer than PEX-168. This indicates that, for the specific sequence of the present invention, the branched PEG is more suitable for the sequence of the present invention from the long-acting effect of the modification, and the large molecular weight PEG is more suitable for the sequence of the present invention.

Experimental Example 3: Acute toxicity test of subcutaneous injection in mice

1. Test drugs: PEX166, PEX168, PEX169, PEX170.

Storage method: Keep away from light and store at -20 °C.

Preparation method: Weigh different amounts of the above compounds, completely dissolve and dilute with PEX special solvent, prepare 50mg/ml PEX166 colorless transparent solution, 100mg/ml PEX168, PEX 170 colorless transparent solution and 150mg/ml PEX 169 colorless transparent solution (the molar concentration of the above solution is equal:).

Route and volume of administration: A single subcutaneous injection is administered at a volume of 25 ml/kg.

2. Test method

Kunming mice (SPF grade:) in each test compound group were 10 males and 10 females, and the mice were poisoned and died within 14 days after administration. Toxic reactions focus on symptoms, duration, duration of toxicity, duration, and recovery time. The animals were weighed into the room, d0 (before administration), and dl~dl4.

3. Test results

During the administration, after administration, and until the end of the observation period, none of the mice died, and they were all free to move, and no abnormalities were observed in coat color, feces, and other conditions.

Weight: The results are shown in Table 3.

After PEX166 is administered 241! ~ 96h (dl~d4), the weight of both male and female mice decreased significantly, reached the lowest point at d4, and then recovered quickly, and returned to the pre-dose weight level at d7.

After PEX168 is administered 241! ~ 48h (dl~d2), the weight of both male and female mice decreased significantly, reached the lowest point at d2, and then recovered quickly, and returned to the pre-dose weight level at d5.

PEX169 after administration 241! ~ 120h (dl~d5), the weight of both male and female mice decreased significantly, reached the lowest point at d5, then gradually recovered, and returned to the pre-dose weight level until dlO.

After PEX170 administration 241! ~ 72h (dl~d3), the weight of both male and female mice decreased significantly, reached the lowest point at d3, then gradually recovered, and returned to the pre-dose weight level at d9.

Anatomy: On day 14 after the mice were sacrificed, no abnormalities were observed by necropsy. Table 3 Mean body weight change in mice with acute toxicity test by subcutaneous injection (g) Compound dO dl d2 d3 d4 d5 d6 d7 d8 d9 dlO dl l dl2 dl3 dl4

PEX168 20.0 17.9 17.1 17.8 19.0 20.3 20.9 21.8 22.7 23.9 25.2 26.1 27.9 29.1 30.2

PEX169 19.9 18.3 17.7 16.9 16.3 15.7 16.6 17.2 18.3 19.1 20.0 21.3 22.6 24.0 25.5

PEX170 20.5 18.3 17.1 16.3 16.9 17.4 18.2 19.0 19.8 20.7 21.9 23.2 24.7 26.1 27.5

Conclusion: A single subcutaneous injection of PEX 166~PEX170 (equivalent amount) has significant inhibitory effect on the food intake and body weight of mice. This inhibition can be gradually restored with the withdrawal of drugs. The inhibition of PEX 168 is weaker than that of the other three. Compounds.

Claims

Claims:

A branched PEG-modified GLP-1 analogue and a pharmaceutically acceptable salt thereof, wherein the structure of the GLP-1 analogue is as shown in formula ( ):

HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC-NH ₂

(1).

2. The branched PEG-modified GLP-1 analogue according to claim 1 and a pharmaceutically acceptable salt thereof, wherein the branched PEG is of a two-branched type.

3. The branched PEG-modified GLP-1 analogue according to claim 1 and a pharmaceutically acceptable salt thereof, wherein the linker of the branched PEG is Lys.

The branched PEG-modified GLP-1 analogue according to claim 1, wherein the branched PEG has a molecular weight of 20 to 80 kD, preferably 40 kD, and a pharmaceutically acceptable salt thereof.

The branched PEG-modified GLP-1 analogue according to any one of claims 2 to 4, wherein the branched PEG is mPEG2-Lys-MAL, and a pharmaceutically acceptable salt thereof.

The branched PEG-modified GLP-1 analogue according to claim 5, wherein the structure is as shown in the formula (Π):

His-D-Ala ~ Glu- Gly- Thr- Phe- Thr- Ser- Asp- Leu- Ser- Lys

— Gin — Nle — Glu — Glu — Glu — Ala — Val — Arg — Leu — Phe — lie — Glu

— Trp — Leu — Lys — Gin — Gly — Gly — Pro — Ser — Ser — Gly — Ala — Pro

A method for producing a branched PEG-modified GLP-1 analogue according to any one of claims 1 to 6 and a pharmaceutically acceptable salt thereof, which comprises a method of synthesis, purification and drying.

8. The method according to claim 7, wherein the synthesis method is selected from a solid phase or a liquid phase method.

9. The method of claim 7, wherein the purification method is selected from the group consisting of reverse phase high performance liquid phase, ion exchange or gel filtration purification methods.

10. The method according to claim 7, wherein the drying method is freeze drying.

Use of a branched PEG-modified GLP-1 analogue according to any one of claims 1 to 6 and a pharmaceutically acceptable salt thereof for the treatment and/or prevention of type 2 diabetes.