METHOD FOR MODIFICATION OF POLYPETIDE AND USES

Abstract

Provided are a method for the modification of a polypeptide and uses. The method comprises the following steps: (1) introducing an X into the N-terminus of a polypeptide, thereby obtaining X-polypeptide; (2) oxidizing the X into an aldehyde group; (3) adding a reducing agent, and covalently coupling the oxidation product obtained in step (2) with PEG, thereby obtaining a PEG-modified polypeptide, wherein X is threonine or serine. In the present application, a single component of PEG-modified polypeptide is obtained by introducing a threonine or serine into the N-terminus of the polypeptide, and deriving the amino alcohol structure at the ortho-position of the N-terminus of the polypeptide as an aldehyde group by using a high-specificity oxidation method and covalently coupling the aldehyde group with PEG. The method has a strong universality and a wide range of application, and the method for separating the modified polypeptide is simple and convenient, thereby improving the stability and the circulating half-life of the polypeptide.

Claims

1. A method for modification of a polypeptide, comprising the following steps: (1) introducing an X to the N-terminus of a polypeptide to obtain an X-polypeptide; (2) oxidizing the X into an aldehyde group; and (3) adding a reducing agent, and covalently coupling the oxidation product obtained in step (2) with polyethyleneglycol (PEG) to obtain a PEG-modified polypeptide; wherein X is threonine or serine.

2. The method according to claim 1, wherein the method of the introducing in step (1) comprises a solid-phase synthesis method or a biological expression method.

3. The method according to claim 2, wherein the solid-phase synthesis method is a Fmoc method.

4. The method according to claim 2, wherein the biological expression method comprises transforming a constructed X-polypeptide expression vector into host bacteria, inducing and collecting the bacteria, and performing lysing and purification to obtain the X-polypeptide.

5. The method according to claim 1, wherein the oxidizing in step (2) is carried out with an oxidizing agent; preferably, the oxidizing agent comprises a periodate, preferably sodium periodate; preferably, the molar ratio of the oxidizing agent to the X-polypeptide is (1-3):1; preferably, the oxidizing in step (2) is carried out at a temperature of 3° C. to 6° C., preferably 3° C. to 4° C.; preferably, the oxidizing in step (2) is carried out for 20 minutes to 40 minutes, preferably 30 minutes to 35 minutes.

6. The method according to claim 1, wherein the reducing agent in step (3) comprises any one or a combination of at least two of sodium borohydride, sodium borohydride acetate or sodium cyanoborohydride, preferably sodium cyanoborohydride.

7. The method according to claim 1, wherein the PEG in step (3) is methoxypolyethylene glycol; preferably, an end group of methoxypolyethylene glycol in step (3) comprises any one of an amino group, an oxyamino group or hydrazide; preferably, the molar ratio of the PEG to the oxidation product in step (3) is (4-6):1; preferably, the covalently coupling in step (3) is carried out at a temperature of 3° C. to 6° C., preferably 3° C. to 4° C.; preferably, the covalently coupling in step (3) is carried out for 1 hour to 3 hours; preferably, the covalently coupling in step (3) is carried out at a pH of 4 to 5, preferably 4 to 4.5.

8. The method according to claim 1, comprising the following steps: (1) introducing an X to the N-terminus of a polypeptide in a solid-phase synthesis method or a biological expression method to obtain an X-polypeptide; (2) adding a periodate oxidizing agent at a molar ratio of the oxidizing agent to the X-polypeptide of (1-3):1, and reacting for 20 minutes to 40 minutes at 3° C. to 6° C., to oxidize the X to an aldehyde group; and (3) adding a reducing agent, and covalently coupling the oxidation product obtained in step (2) with methoxypolyethylene glycol at 3° C. to 6° C. at pH of 4 to 5 for 1 hour to 3 hours, wherein the molar ratio of the methoxypolyethylene glycol to the oxidation product is (4-6):1, to obtain a PEG-modified polypeptide; wherein X is threonine or serine.

9. A polypeptide analog prepared by the method according to claim 1.

10. A GLP-1 receptor agonist analog prepared by the method according to claim 1.

11. The GLP-1 receptor agonist analog according to claim 10, wherein the GLP-1 receptor agonist analog has a structure of PEG-X-GLP-1 receptor agonist; wherein X is threonine or serine.

12. The GLP-1 receptor agonist analog according to claim 10, wherein the GLP-1 receptor agonist comprises any one of GLP-1, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, benaglutide or semaglutide; preferably, the PEG is methoxypolyethylene glycol; preferably, an end group of methoxypolyethylene glycol comprises any one of an amino group, an oxyamino group or hydrazide; preferably, the molecular weight of methoxypolyethylene glycol is 2000 Da to 50000 Da, preferably 5000 Da to 20000 Da, further preferably 5000 Da to 10000 Da.

13. A pharmaceutical composition, comprising the polypeptide analog according to claim 9.

14. The pharmaceutical composition according to claim 13, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

15. (canceled)

16. A method for preventing and/or treating obesity, diabetes or Alzheimer's disease, comprising administering an effective amount of the GLP-1 receptor agonist analog according to claim 10 to subject in need thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 is an SDS-PAGE electropherogram of T-GLP-1, where 1 represents standard protein samples whose molecular weights are 14.4 KDa, 18.4 KDa, 25.0 KDa, 35.0 KDa, 45.0 KDa, 66.2 KDa, and 116.0 KDa in sequence, 2 represents a T-GLP-1-containing mixture of E. coli soluble expression, 3 represents T-GLP-1 initially purified by affinity chromatography, and 4 represents T-GLP-1 obtained by ion-exchange chromatography; and

[0063] FIG. 2 is a diagram of high performance liquid chromatography (HPLC) of T-GLP-1, T-GLP-1, and mPEG.sub.5k-T-GLP-1.

DETAILED DESCRIPTION

[0064] To further elaborate the technical means adopted and the effects achieved in the present application, the present application is described below in conjunction with the examples and drawings. It is to be understood that the specific examples set forth below are intended to illustrate but not to limit the present application.

[0065] Experiments without specific techniques or conditions noted in the examples are conducted according to techniques or conditions described in the literature in the art or a product specification. The reagents or instruments used herein without manufacturers specified are conventional products commercially available from proper channels.

Example 1 Synthesis of X-GLP-1 Receptor Agonist by Fmoc Solid-Phase Method

[0066] The activated Fmoc-rink amide-MBHA resin was placed in a CS Bio polypeptide synthesizer and was connected to amino acids according to the sequence of the X-GLP-1 receptor agonist through the deprotection and coupling steps sequentially to obtain a resin to which the X-GLP-1 receptor agonist was connected. The efficiency of the coupling step was measured by the ninhydrin method, and if the color reaction was negative, go to the next coupling cycle. After the completion of the reaction, the polypeptide was lysed from the resin by adding a lysis buffer, and a crude X-GLP-1 receptor agonist was obtained after washing. The crude product was purified by preparative HPLC, the mobile phase was A (water+0.1% TFA) and B (acetonitrile+0.1% TFA), the target peak was collected, and the crude product was freeze-dried to obtain the pure X-GLP-1 receptor agonist.

[0067] In this example, X is threonine or serine, and the GLP-1 receptor agonist is GLP-1, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, benalglutide or semaglutide.

Example 2 Biological Expression of X-GLP-1 Receptor Agonist

[0068] A threonine codon or a serine codon was added to the end of a GLP-1 receptor agonist gene fragment and constructed to the upstream of the enterokinase site (DDDDK) and the histidine tag in the pET28a vector to obtain an expression plasmid of the fused peptide Histag-DDDK-T-GLP-1. The constructed plasmid was transformed into Escherichia coli BL21 (Takara) and induced with 0.5 mM IPTG, and the bacteria were collected. After the bacteria were lysed, the supernatant was collected by centrifugation and initially purified using a Ni affinity column, then the enterokinase site sequence and histidine tag were removed by enterokinase digestion, and finally, the supernatant was purified using ion-exchange chromatography to obtain the X-GLP-1 receptor agonist.

[0069] In this example, X is threonine or serine, and the GLP-1 receptor agonist is GLP-1, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, benalglutide or semaglutide.

[0070] The sample during the expression and purification process was collected for SDS-PAGE detection. As shown in FIG. 1, for example, the purity of the obtained T-GLP-1 was more than 95%.

Example 3 N-Terminal Site-Directed Oxidation of X-GLP-1 Receptor Agonists

[0071] The X-GLP-1 receptor agonist was dissolved in the PB buffer (50 mM, pH=7.0), 1 mg/mL NaIO.sub.4 solution was added at the molar ratio of NaIO.sub.4 to the X-GLP-1 receptor agonist of 2:1, the reaction was carried out at 4° C. for 30 min, and 100 μL of ethylene glycol was added to terminate the reaction.

[0072] The sample was added to a GE G25 desalination column, the polypeptide peak was collected, and an achromatic magenta color reagent was added for color development.

[0073] It was found from color development results that the oxidized X-GLP-1 receptor agonist was bright red, a feature color of the aldehyde group in the achromatic magenta detection, indicating that NaIO.sub.4 oxidized the threonine or serine to the aldehyde group.

[0074] In this example, X is threonine or serine, and the GLP-1 receptor agonist is GLP-1, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, benalglutide or semaglutide.

[0075] The samples before and after oxidation were subjected to high performance liquid chromatography. The detection results were shown in FIG. 2. For example, the retention time of T-GLP-1 on the C18 column was 14.68 min, and the retention time of T-GLP-1-CHO formed after oxidation with the unique aldehyde group was 15.01 min.

Example 4 Preparation of mPEG.SUB.5k.-HZ-Modified GLP-1 Analog

[0076] The mPEG hydrazide powder (mPEG.sub.5k-HZ) with a molecular weight of 5000 Da was added to the oxidized Thr-GLP-1, where the molar ratio of mPEG.sub.5k-HZ to Thr-GLP-1 was 5:1. 5 mM of sodium cyanoborohydride was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 4.5 for 2 h at 4° C.

[0077] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the mobile phase was a Na.sub.2SO.sub.4 buffer system (0.1 M, pH=7.4) containing 20 mM of PB at a flow rate of 0.6 mL/min. The peak was collected at a detection wavelength of 220 nm. The collected sample was dialyzed overnight in a system containing 20 mM of PB and 5% mannitol and then stored after ultrafiltration and concentration.

[0078] Results are shown in FIG. 2, and the retention time of the successfully separated and purified mPEG.sub.5k-T-GLP-1 reached 15.3 min, with an overall modification rate of 80.3%.

Example 5 Preparation of mPEG.SUB.20k.-HZ-Modified Exenatide Analog

[0079] The mPEG hydrazide powder (mPEG.sub.20k-HZ) with a molecular weight of 20000 Da was added to the oxidized Thr-exenatide, where the molar ratio of mPEG.sub.20k-HZ to Thr-exenatide was 5:1. 5 mM of sodium cyanoborohydride was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 4.5 for 2 h at 4° C.

[0080] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the mobile phase was a Na.sub.2SO.sub.4 buffer system (0.1 M, pH=7.4) containing 20 mM of PB at a flow rate of 0.6 mL/min. The peak was collected at a detection wavelength of 220 nm. The collected sample was dialyzed overnight in a system containing 20 mM of PB and 5% mannitol and then stored after ultrafiltration and concentration.

[0081] It was found from the results that mPEG.sub.20k-HZ-modified exenatide was successfully separated and purified.

Example 6 Preparation of mPEG.SUB.10k.-O—NH.SUB.2.-Modified Liraglutide Analog

[0082] The mPEG oxyammonia powder (mPEG.sub.10k-O—NH.sub.2) with a molecular weight of 10000 Da was added to the oxidized Thr-liraglutide, where the molar ratio of mPEG.sub.10k-O—NH.sub.2 to Thr-liraglutide was 5:1. 5 mM of sodium cyanoborohydride was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 4.5 for 2 h at 4° C.

[0083] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the collected sample was detected by HPLC. It was found that the mPEG.sub.10k-O—NH.sub.2-modified liraglutide was successfully separated and purified.

Example 7 Preparation of mPEG.SUB.2k.-NH.SUB.2.-Modified Albiglutide Analog

[0084] The mPEG amino powder (mPEG.sub.2k-NH.sub.2) with a molecular weight of 2000 Da was added to the oxidized Ser-albiglutide, where the molar ratio of mPEG.sub.2k-NH.sub.2 to Ser-albiglutide was 6:1. 5 mM of sodium borohydride was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 4 for 3 h at 3° C.

[0085] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the collected sample was detected by HPLC. It was found that the mPEG.sub.2k-NH.sub.2-modified albiglutide was successfully separated and purified.

Example 8 Preparation of mPEG.SUB.50k.-NH.SUB.2.-Modified Dulaglutide Analog

[0086] The mPEG amino powder (mPEG.sub.50k-NH.sub.2) with a molecular weight of 50000 Da was added to the oxidized Ser-dulaglutide, where the molar ratio of mPEG.sub.50k-NH.sub.2 to Ser-dulaglutide was 4:1. 5 mM of sodium borohydride acetate was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 5 for 1 h at 6° C.

[0087] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the collected sample was detected by HPLC. It was found that the mPEG.sub.50k-NH.sub.2-modified dulaglutide was successfully separated and purified.

Example 9 Preparation of mPEG.SUB.5k.-HZ-Modified Semaglutide Analog

[0088] The mPEG hydrazide powder (mPEG.sub.5k-HZ) with a molecular weight of 5000 Da was added to the oxidized Thr-semaglutide, where the molar ratio of mPEG.sub.5k-HZ to Thr-semaglutide was 5:1. 5 mM of sodium cyanoborohydride was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 4.5 for 2 h at 4° C.

[0089] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the collected sample was detected by HPLC. It was found that the mPEG.sub.50k-NH.sub.2-modified semaglutide was successfully separated and purified.

Example 10 Preparation of mPEG.SUB.5k.-HZ-Modified Thymosin Analog

[0090] The mPEG hydrazide powder (mPEG.sub.5k-HZ) with a molecular weight of 5000 Da was added to the oxidized Ser-thymosin, where the molar ratio of mPEG.sub.5k-HZ to Ser-thymosin was 5:1. 5 mM of sodium cyanoborohydride was added as a reducing agent during the reaction, and the oscillatory reaction was carried out at pH of 4.5 for 2 h at 4° C.

[0091] After the completion of the reaction, the reaction solution was subjected to chromatographic separation on the GE Superdex 75 10/300 GL column, and the collected sample was detected by HPLC. It was found that the mPEG.sub.50k-NH.sub.2-modified thymosin was successfully separated and purified.

Example 11 In Vivo Pharmacokinetics Detection of mPEG.SUB.5k.-T-GLP-1

[0092] The in vivo pharmacokinetic activity detection of mPEG.sub.5k-T-GLP-1 was carried out with 30 male SD rats, aged 7-8 weeks, weighing 200-250 g/rat. These rats were randomly grouped and injected with GLP-1 or mPEG.sub.5k-T-GLP-1 at a dose of 2 μg/kg by weight, and the injection method was subcutaneous injection.

[0093] Blood was taken from the eye socket of each rat at 1, 2, 4, 8, 30, 60, 120 and 240 min, timed from the first injection, then placed in tubes containing EDTA and centrifuged at 5000 rpm for 5 min at 4° C., the cells of the lower layer were discarded, and the supernatant was stored at −80° C.

[0094] The stored samples were all taken out and thawed. The GLP-1 concentration at different time points was measured using the rat GLP-1 enzyme-linked immunoassay ELISA testing kit, and the half-life and AUC value of GLP-1 before and after PEG modification were calculated based on the measurement results.

[0095] The stored samples were all taken out and thawed. The GLP-1 concentration at different time points was measured using the rat GLP-1 enzyme-linked immunoassay ELISA testing kit, and the half-life and AUC value of GLP-1 before and after PEG modification were calculated based on the measurement results.

[0096] The results show that the half-life of mPEG.sub.5k-T-GLP-1 in vivo is increased by more than 60-fold, and the AUC value is 10-fold higher than that of the original GLP-1.

Example 12 In Vivo Pharmacodynamics Activity Assay of mPEG.SUB.5k.-T-GLP-1

[0097] The in vivo pharmacodynamics activity detection of mPEG.sub.5k-T-GLP-1 was carried out with 30 type II diabetic db/db mice, aged 7-8 weeks, weighing 8-250 g/mouse. These mice were fed a high-fat diet and made into models. These mice were observed daily from the start of animal feeding. Each of the mice was weighed every Wednesday after 8 hours of fasting, and meanwhile, the tail vein blood glucose levels of the mice were measured.

[0098] After 8 weeks of intervention, 18 mice were randomly selected and then divided into three groups. Each of these 18 mice was injected with GLP-1 or mPEG.sub.5k-T-GLP-1 at a dose of 2 μg/kg by weight, and the injection method was subcutaneous injection.

[0099] Blood was taken from the tail vein of each mouse at 1, 2, 4, 8, 30, 60, 120 and 240 min, timed from the first injection, then placed in tubes containing EDTA and centrifuged at 5000 rpm for 5 min at 4° C., the cells of the lower layer were discarded, and the supernatant was stored at −80° C.

[0100] The stored samples were all taken out and thawed, and the blood glucose concentration and insulin content were measured using the enzyme-linked immunoassay kit.

[0101] The results show that mPEG.sub.5k-T-GLP-1 had a significant hypoglycemic effect in vivo, and after 5 hours after injection, the blood glucose of db/db mice reached the normal level.

[0102] In summary, in the present disclosure, a PEG-modified polypeptide with a single component is obtained by introducing a threonine or serine into the N-terminus of the polypeptide by using a solid-phase synthesis method or a biological expression method, deriving the amino alcohol structure at the ortho-position of the N-terminus of the polypeptide as an aldehyde group by using a high-specificity oxidation method, and covalently coupling the aldehyde group with PEG. The method has a robust universality and a wide range of application, and the method for separating the modified polypeptide is simple and convenient, thereby improving the stability and the circulating half-life of the polypeptide. The GLP-1 receptor agonist analog prepared by the method for modification of a polypeptide of the present application has 100% homology with the GLP-1 receptor agonist, thereby preserving the hypoglycemic effect of the GLP-1 receptor agonist and avoiding problems of immunogenicity and drug resistance. The introduced threonine or serine effectively prevents the N-terminal His-Ala sequence of GLP-1 from being degraded by dipeptidyl peptidase IV, and the half-life of the obtained GLP-1 analog in vivo is increased by more than 60-fold, and the AUC value is 10-fold higher than that of the original GLP-1.

[0103] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.