Exendin or its analogs with polyethylene group and its preparation and application

A polyethylene glycol group, polyethylene glycol technology, applied in the field of modified Exendin or its analogues, can solve the problems of impractical application, increased dosage, and decreased biological activity

Active Publication Date: 2008-02-20
SHANGHAI HUAYI BIO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is that the time interval after Exendin or its analogs are used in the body after a single administration is relatively short, and long-term injection will cause antibodies to be produced in the body. The biological activity of existing polyethylene glycol-modified Exendin or its analogs is severely reduced, so that the dosage is greatly increased, and it cannot be practically applied

Method used

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  • Exendin or its analogs with polyethylene group and its preparation and application
  • Exendin or its analogs with polyethylene group and its preparation and application
  • Exendin or its analogs with polyethylene group and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The solid-phase chemical synthesis of embodiment 1 Exendin-4 analog

[0064] This embodiment introduces the synthesis of Exendin-4 analogs with the following structure by the method of solid-phase chemistry:

[0065] [His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Arg-Glu-Glu-Glu-Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH] (SEQ ID NO: 232).

[0066] (1) Amino acid monomers used

[0067] Fmoc-L-Ala-OH

Fmoc-L-Lys(Boc)-OH

Fmoc-L-Asn(Trt)-OH

Fmoc-L-Met-OH

[0068] Fmoc-L-Asp(OtBu)-OH

Fmoc-L-Phe-OH

Fmoc-L-Gln(Trt)-OH

Fmoc-L-Pro-OH

Fmoc-L-Glu(OtBu)-OH

Fmoc-L-Ser(tBu)-OH

Fmoc-L-Gly-OH

Fmoc-L-Thr(tBu)-OH

Fmoc-L-His(Trt)-OH

Fmoc-L-Trp-OH

Fmoc-L-Ile-OH

Fmoc-L-Tyr(tBu)-OH

Fmoc-L-Leu-OH

Fmoc-L-Val-OH

[0069] The abbreviation in the above formula means:

[0070] Fmoc: 9-fluorenylmethoxycarbonyl

[0071] BOC: ...

Embodiment 2

[0093] The preparation of the genetic engineering method of embodiment two Exendin-4 analogs

[0094] This embodiment introduces the synthesis of the Exendin-4 analog with the following structure by the method of genetic engineering:

[0095] [His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Leu-Glu-Glu-Glu-Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Arg-OH]

[0096] (SEQ ID NO: 251).

[0097] A. According to the amino acid sequence of this Exendin-4 analog, synthesize the gene fragment:

[0098] (1) 5'AAT TCC ATG CAC GGC GAA ACC TTC ACC AGC GAT CTG AGC AAA CAG CTG GAA GAA GAAGCG GTT AA (SEQ ID NO: 266)

[0099] (2) 5' ACTG TTC ATC GAA TGG CTG AAA AAC GGC GGC CCG AGC AGC GGC CCG CCG CCG CCG AGCCGT TAG A (SEQ ID NO: 267)

[0100] (3) 5' AGCTT CTA ACG GCT CGG CGG CGG CGC GCT GCT CGG GCC GCC GTT TTT CAG CCA TTC GATGA (SEQ ID NO: 268)

[0101] (4) 5' ACAG TTT AAC CGC TTC TTC TTC CAG CTG TTT GCT CAG ATC GCT GGT GAA GGT GCC TTCGCC...

Embodiment 3

[0116] Example 3 Modification of Exendin-4 with linear monomethoxypolyethylene glycol (molecular weight=5,000 Daltons)

[0117] Weigh 1.0 mg Exendin-4 into each test tube, three test tubes in total. Dissolve in phosphate buffer solutions with different pH values, and then add 5.8 mg of N-hydroxysuccinimide-activated monomethoxypolyethylene glycol (molecular weight = 5,000 Daltons) to each test tube, place on a shaker, React at room temperature for 1 hour. Then use Agilent 1100 series chromatograph (Agilent 1100) to analyze samples (analytical conditions are: adopt 0.1% phosphoric acid mobile phase A and 0.1% phosphoric acid+80% acetonitrile mobile phase B, gradient is: 35-70% mobile phase B / 25 minutes ), to detect the amount of Exendin-4 not modified by monomethoxypolyethylene glycol. The results are as follows:

[0118] Table: Effects of different pH on PEGylation of Exendin-4

[0119] pH value

[0120] 8.5

[0121] Using a rotary evaporator to remov...

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Abstract

An Exendin with carbowax group or the analog of Extendin and the preparation of Extendin relates to the technical field of Exendin or the analog of Extendin which is modified with one or more than one carbowax and the preparation of the Extendin. The present invention aims at solving the technical problem that the medicine taking interval is short; the bioactivity is greatly reduced after being modified with carbowax so as to increase the medicament dose, which yet cannot be carried out practically. The Extendin or the analog of Extendin of the present invention which is modified with carbowax is characterized by long hall-life, high activity and low immunogenicity.

Description

technical field [0001] The present invention relates to a modified Exendin or its analogues, in particular to a single or multiple polyethylene glycol modified Exendin or its analogues and its preparation, administration method and application. Background technique [0002] Glucagon-like peptide-1 was first identified by researchers in 1987 as a glucose-dependent gut secretory hormone peptide. This polypeptide transmits signals through G protein-coupled receptors on cells, thereby stimulating β islet cells to secrete insulin, inhibiting glucagon secretion, inhibiting gastric emptying and gastric acid secretion and other physiological effects. [0003] Exendin is a class of polypeptides (J.Biol.Chem.1990, 265, 20259-20262; J.Biol.Chem.1992, 267, 7402-7405) found in the saliva of the monster monster and bead monster. This type of peptide represented by Exendin-4 has high homology with glucagon-like peptide-1 [7-36]. Studies have found that Exendin can also bind to the glucag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K38/26A61P3/10
CPCC07K14/57563A61K47/48215A61K38/00A61K47/48338A61K38/26A61K47/60A61K47/65A61P3/10A61P3/06A61P5/50A61K47/50A61K38/22C07K14/575C07K17/06
Inventor 包文超徐宏景余刚左亚军
Owner SHANGHAI HUAYI BIO LAB CO LTD
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