WO2022266927A1 - Liraglutide variant, preparation method therefor, and application thereof - Google Patents

Abstract

Disclosed are a liraglutide variant, a preparation method therefor, and an application thereof. The twelfth amino acid Ser of the liraglutide variant is allosterically regulated to a D-type amino acid. The biological activity of the liraglutide variant provided by the present invention is twice that of the liraglutide, and the liraglutide variant can be used for preparing hypoglycemic and weight loss drugs.

Description

A variant of liraglutide and its preparation method and application technical field

The invention relates to the technical field of polypeptides, in particular to a variant of liraglutide and its preparation method and application.

Background technique

Liraglutide (liraglutide) was developed by Novo Nordisk in Denmark. It was first launched in the European Union in July 2009, and it was launched in Japan and the United States in 2010. Belonging to the glucagon-like peptide-1 (GLP-1) receptor agonist hypoglycemic drugs, it can stimulate the secretion of natural insulin. Its molecular structure is similar to that of GLP-1, the difference is that liraglutide changes the 34th Lys of GLP-1 to Arg, and adds a palmitoyl fatty acid side chain at the 26th position.

Peptide drugs are easily degraded by proteases in the body, and liraglutide is also easily degraded by DPP-IV enzymes in the human body, resulting in a decrease in bioavailability. As an injection, liraglutide needs to be injected once a day, which brings great psychological and economic burdens to patients. Therefore, in order to improve medication compliance, it is necessary to modify its structure or develop new dosage forms to improve its biological activity or bioavailability.

Contents of the invention

In view of this, the purpose of the present invention is to provide a variant of liraglutide and its preparation method and application. The biological activity of the liraglutide variant is significantly improved, which is twice that of the original liraglutide preparation.

The present invention provides a variant of liraglutide, the 12th amino acid L-Ser is modified into D-Ser, and the amino acid sequence is shown in SEQ ID NO: 1:

H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N -α-Palmitoy-L-γ-Glutamyl-))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

The relative potency of the liraglutide variant provided by the present invention is 204% relative to the original research reference preparation, indicating that its biological activity is twice that of liraglutide, and it can be used to prepare hypoglycemic and weight loss drugs and has broad prospects .

The present invention also provides the application of the liraglutide variant in the preparation of hypoglycemic drugs.

And, the application of the liraglutide variant in the preparation of weight-loss drugs.

The present invention also provides a preparation method of the liraglutide variant, comprising the following steps:

Step 1: Take Fmoc-Gly-resin and add it to the solid-phase reaction column, and swell it with DMF for 30 minutes; weigh the N-terminal protected amino acid, dissolve it in DMF, add a coupling reagent to activate it, add it to the reaction column, and react 1-3 Hour;

Step 2: extract the reaction solution, remove the Fmoc protecting group with a mixed solution of piperidine and DMF;

Step 3: Repeat the above steps 1 to 2, according to the sequence of the amino acid sequence shown in SEQ ID NO: 1 from the C-terminal to the N-terminal, couple the protected amino acids one by one until the last amino acid in the coupling main chain ends, and obtain the peptide resin;

Step 4: cracking the peptide resin to obtain crude peptides, and purifying the crude peptides to obtain refined peptides.

In the present invention, the type of resin is not particularly limited, and the types commonly used in the field are sufficient. In some embodiments, the resin is Wang resin.

When coupling amino acids one by one, since each amino acid has a protecting group, it is necessary to remove the N-terminal protecting group before coupling. The removal agent used to remove the N-terminal Fmoc protecting group in the present invention is preferably a mixed solution of piperidine and DMF , including but not limited to. In some specific embodiments, 20% piperidine in DMF solution is used to remove the Fmoc protecting group; the times of removal are two times, 5 min and 7 min respectively.

In some embodiments, in step 3, the coupling agent is DIC/Oxymap pure, and the activation time is 3-5 minutes.

In some embodiments, in step 3, the protected amino acid used for coupling the 12th amino acid of liraglutide is Fmoc-D-Ser(tBu)-OH, and the protected amino acid used for the 20th amino acid is Fmoc-Lys(Pal- Glu-OtBu)-OH.

In some embodiments, in step 4, the cracking agent used for cracking is composed of trifluoroacetic acid, purified water, phenol and EDT; the volume ratio of the trifluoroacetic acid, purified water, phenol and EDT is 87.5:5:5 : 2.5.

In step 4 of the present invention, the purification is RP-HPLC purification. The chromatographic columns used for purification include but are not limited to C18 chromatographic columns.

In the variant of liraglutide provided by the present invention, the 12th amino acid Ser is modified into a D-type amino acid, and the amino acid sequence is shown in SEQ ID NO:1. The relative potency of the liraglutide variant provided by the present invention is 204% relative to the original research reference preparation, indicating that its biological activity is twice that of liraglutide, and it can be used to prepare hypoglycemic and weight loss drugs and has broad prospects .

Description of drawings

Fig. 1 shows the dose-response curve graph of biological reference substance (BRPS) and system suitability sample (SSS);

Fig. 2 shows the dose-response curve of biological reference substance (BRPS) and liraglutide variant solution (TS);

Figure 3 shows a comparison curve of hypoglycemic effects of liraglutide and liraglutide variants;

Figure 4 shows the comparison curve of the weight loss effect of liraglutide and liraglutide variants.

detailed description

The present invention provides a variant of liraglutide and its preparation method and application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

The reagents and instruments used in the present invention are all common commercial products and can be purchased in the market.

In the present invention, the meanings of English abbreviations are shown in Table 1.

Table 1

Abbreviation and English	meaning
DMF	N,N-Dimethylformamide
Fmoc	9-Fluorenyloxycarbonyl
DIC	N,N-Diisopropylcarbodiimide
Oxyma Pure	2-Oxime ethyl cyanoacetate
CAMP	cyclic adenosine monophosphate
EDT	1,2-ethanedithiol

PBS buffer	Phosphate Buffered Saline
EDTA	Ethylenediaminetetraacetic acid

In the variant of liraglutide provided by the present invention, the 12th serine is modified into D-type serine, and the amino acid sequence is shown in SED ID NO: 1:

H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-( N-α-Palmitoy-L-γ-Glutamyl-))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

Correspondingly, the present invention provides a preparation method of the liraglutide variant. In some embodiments, the present invention adopts Fmoc solid-phase synthesis strategy, and specific method is as follows:

(1) Add Fmoc-Gly-Wang Resin resin into the solid phase reaction column, and swell with DMF for 30 minutes;

(2) Weigh the N-terminal protected amino acid, dissolve it in DMF, add the coupling reagent DIC/Oxymap pure, activate for 3-5 minutes, put it into the reaction column, and react for 1-3 hours. The reaction was monitored with ninhydrin and the resin was clear, indicating complete reaction.

(3) Extract the reaction liquid, remove the Fmoc protection twice with 20% (V/V) piperidine DMF mixed solution, the time is 5 minutes and 7 minutes respectively, detect the reaction with ninhydrin, and require the resin to develop color.

(4) Repeat the above steps, wherein the 12th amino acid is coupled with Fmoc-D-Ser(tBu)-OH, and the 20th amino acid is coupled with Fmoc-Lys(Pal-Glu-OtBu)-OH until the coupling is completed in the main chain all amino acids.

(5) After the obtained peptide resin is shrunk and dried, the synthesized fully protected peptide resin is cracked with TFA for 2-4 hours, the lysate is precipitated with ether, centrifuged, dried and other steps are taken to obtain the crude peptide. Finally, the crude peptide was prepared and purified with a C8 chromatographic column to obtain the refined peptide.

Below in conjunction with embodiment, further set forth the present invention:

Example 1: Solid phase synthesis of liraglutide variants

Take Fmoc-Gly-Wang Resin resin (50.0g) with a degree of substitution of 0.3mmol/g, add it to the solid-phase reaction column, wash it twice with DMF (300mL), then swell with DMF (300mL) for 30 minutes; weigh the N-terminal protected amino acid (3eqv), dissolved in DMF (300mL), added coupling reagent DIC (3.9qev)/Oxymapure (3.6eqv), activated for 3-5 minutes, added to the reaction column, and reacted for 1-3 hours. The reaction was monitored with ninhydrin and the resin was clear, indicating complete reaction. The reaction solution was extracted, and the Fmoc protection was deprotected twice with 20% (V/V) piperidine DMF mixed solution (300mL) for 5 minutes and 7 minutes respectively, and the ninhydrin detection reaction required the resin to develop color. Repeat the above steps, wherein the 12th amino acid is coupled with Fmoc-D-Ser(tBu)-OH (3eqv), and the 20th amino acid is coupled with Fmoc-Lys(Pal-Glu-OtBu)-OH (3eqv) until the coupling is completed All amino acids in the main chain, methanol shrinkage, each dosage (300mL), 127.1g of the peptide resin obtained after drying, the synthetic fully protected peptide resin with (trifluoroacetic acid: purified water: phenol: EDT = 87.5: 5: 5:2.5, v/v) lysis for 2-4 hours, the lysate was precipitated with ether, precipitation, centrifugation, drying and other steps to obtain 62.1 g of crude peptide, and 9.8 g of refined peptide was obtained by reverse phase preparation and purification.

Embodiment 2: biological activity assay experiment

Preparation of sample solvent: Take 1.42g of disodium hydrogen phosphate dihydrate, 14g of propylene glycol, and 5.5g of phenol, add water to dissolve to 1L, adjust the pH to 8.15 with 1mol/L sodium hydroxide solution, and filter through 0.2μm.

Preparation of the liraglutide variant solution: Dissolve the liraglutide variant in a solvent to a concentration of 6 mg/ml to obtain a liraglutide variant solution. The liraglutide variant solution was serially diluted to 10 concentration points (from 3.75 nM to 0.0018 nM) using assay buffer. Each diluted sample was analyzed for cAMP content in duplicate wells, one cell negative control sample and one cell basal level sample were analyzed.

cAMP standard curve: 8 concentration points were prepared from the theoretical cAMP concentration of the standard curve: 0.35nM, 0.69nM, 1.39nM, 2.78nM, 11.13nM, 44.5nM, 89.00nM, 178.00nM. Concentration-response data (DeltaR) were fitted with a 4-parameter logistic model to obtain a cAMP standard curve.

The original preparation of liraglutide (JS68L85) was used as the system suitability sample (SSS), and the original preparation of liraglutide (JS68L68) was used as the biological reference sample (BRPS). Biological reference substance (BRPS), system suitability sample (SSS) and liraglutide variant solution (TS) were serially diluted with analysis buffer to 10 concentration points (7.5nM—3.7×10 ^-3 nM ; 2× compound), each concentration was paralleled to 2 wells, sample dilution was carried out at room temperature, and the reaction was carried out within one hour.

Cell culture: HEK293-GLP1R cells were provided by Shanghai Heyuan Biotechnology Co., Ltd. for cell resuscitation and subculture for 2-3 days. The cells in the culture medium were taken out from the incubator and the cell confluence was visually inspected under a microscope (the cells accounted for 2% of the culture flask). area) is greater than 80% (cell generation is not greater than P15) can be used in the experiment; transfer the cultured cells into a biological safety cabinet, open the cell culture medium bottle, suck out the culture solution with a pipette gun and discard it. In the culture flask (25 cm ² ), 2-3 cm of PBS buffer was added to clean the bottom of the cell culture flask, the PBS solution was taken out, and 1-2 mL of digestion solution (trypsin-0.25% EDTA) was added. When it is observed under a microscope that the surrounding cells become clear and tend to become round, add medium to stop digestion and suspend the cells, and use a centrifuge to centrifuge the cell suspension at 1000 rpm for 4 minutes at room temperature, and remove the supernatant. Add analysis buffer to resuspend the cells, use a cell counter to count the resuspended cells, and then dilute the resuspended cell solution according to the counting results to prepare the cell solution for the experiment (the cell concentration is about 4×10 ⁵ /mL ; Cell viability is above 95%).

HEK293-GLP1 cells were induced to produce cAMP by comparing liraglutide API solution (ie test sample TS), the amount of cAMP produced was represented by the signal ratio of 665nM/620nM, and a curve was fitted by multiple signal ratios of 665nM/620nM , and get the EC50.

The experimental results of the biological reference material (BRPS) and the system suitability sample (SSS) are shown in Figure 1.

The dose-response curves of biological reference substance (BRPS) and liraglutide variant solution (TS) are shown in Figure 2.

It can be seen from Figure 1 that the dose-response curves are parallel, which proves that the system has good adaptability. From the results in Figure 2, it can be seen that

Comparing EC50 of biological reference and liraglutide variant samples for relative potency, calculation of parallelism of dose-response curves and relative potency of liraglutide variant samples for evaluation of liraglutide variants The biological activity of the sample. Relative potency (%)=(EC50 biological reference substance/EC50 test sample or system suitability sample)×100%, where the EC50 value comes from the result of nonlinear fitting in F test. The data were collected, analyzed and organized by BioTek SynergyH1 microplate reader. The experimental results are shown in Table 2.

Table 2

The results showed that the relative potency of the liraglutide variant of the present invention compared with the original reference preparation BRPS was 204%, indicating that the biological activity of the liraglutide variant of the present invention was twice that of liraglutide.

Example 3 Hypoglycemic Effect of Liraglutide Variant

Male Kunming mice (body weight 26-31 g) were randomly divided into 3 groups, 6 mice in each group, without food or water deprivation before the experiment. Equal volumes of normal saline (10 mL/kg), liraglutide (0.6 mg/kg) and liraglutide variant samples (0.3 mg/kg) were injected subcutaneously in a single dose. Blood was collected at the tip of the tail at 0, 1, 4, 8, 12, 14, and 16 hours after the injection, and the blood glucose was tested using the Johnson & Johnson Wenhao blood glucose meter and supporting test strips. Taking the blood glucose values at different time points as the ordinate and time as the abscissa, the time-effect curve of the hypoglycemic effect was established, and the biological half-life of the hypoglycemic effect of liraglutide and liraglutide variants was calculated. As shown in Figure 3, liraglutide variants have comparable biological half-lives to liraglutide. The dose of liraglutide variants was halved, and the hypoglycemic effect was comparable to that of liraglutide, indicating that the hypoglycemic effect was better.

Example 4 Effect of Liraglutide Variant on Body Weight

Fifteen healthy male guinea pigs, each weighing about 300 g, were divided into 5 groups, namely the normal saline group, liraglutide variant and liraglutide. The guinea pigs of the corresponding groups were subcutaneously injected with normal saline (1mL/kg), liraglutide variant (0.6mg/kg), and liraglutide (0.6mg/kg) the next day, with time as the abscissa, The body weight of the guinea pigs was plotted on the ordinate, as shown in Figure 4, during which the body weight of the guinea pigs in the saline group continued to increase. Compared with the normal saline group, the body weight of liraglutide variants and liraglutide guinea pigs given the same dose for two consecutive times decreased by about 7% and 3%, respectively. As a result, it can be seen that given the same dose of liraglutide variant, its effect on reducing the body weight of guinea pigs is stronger than that of liraglutide.

The above are only preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can also be made without departing from the principle of the present invention, and these improvements and modifications should also be considered Be the protection scope of the present invention.

Claims (10)

1. A variant of liraglutide, characterized in that its amino acid sequence is as shown in SEQ ID NO:1.
2. The application of the liraglutide variant described in claim 1 in the preparation of hypoglycemic drugs.
3. The application of the liraglutide variant described in claim 1 in the preparation of weight-loss drugs.
4. The preparation method of liraglutide allomer according to claim 1, is characterized in that, comprises the following steps:

   Step 1: Take Fmoc-Gly-resin and add it to the solid-phase reaction column, and swell it with DMF for 30 minutes; weigh the N-terminal protected amino acid, dissolve it in DMF, add a coupling reagent to activate it, add it to the reaction column, and react 1-3 Hour;

   Step 2: extract the reaction solution, and remove the Fmoc protecting group with a mixed solution of piperidine and DMF;

   Step 3: Repeat the above steps 1 to 2, according to the sequence of the amino acid sequence shown in SEQ ID NO: 1 from the C-terminal to the N-terminal, couple the protected amino acids one by one until the last amino acid in the coupling main chain ends, and obtain the peptide resin;

   Step 4: cracking the peptide resin to obtain crude peptides, and purifying the crude peptides to obtain refined peptides.
5. The preparation method according to claim 4, wherein the resin is 2-chlorotrityl chloride resin or Wang resin.
6. The preparation method according to claim 4, characterized in that in step 2, the Fmoc protecting group is removed with 20% piperidine DMF solution; the number of times of removal is twice, 5 min and 7 min respectively.
7. The preparation method according to claim 4, characterized in that, in step 3, the coupling agent is DIC/Oxymapure, and the activation time is 3-5 minutes.
8. The preparation method according to claim 4, characterized in that, in step 3, the protected amino acid used for coupling the 12th amino acid of liraglutide is Fmoc-D-Ser(tBu)-OH, and the protected amino acid used for the 20th amino acid The protected amino acid is Fmoc-Lys(Pal-Glu-OtBu)-OH.
9. The preparation method according to claim 4, characterized in that, in step 4, the cracking agent used for cracking is composed of trifluoroacetic acid, purified water, phenol and EDTA; the trifluoroacetic acid, purified water, phenol and EDTA The volume ratio is 87.5:5:5:2.5.
10. The method according to any one of claims 1-9, characterized in that, in step 4, the purification is RP-HPLC purification.

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