Exendin-4 analogue pegylated with polyethylene glycol or derivative thereof, preparation method thereof, and pharmaceutical compostion for preventing or treating diabetes, containing same as active ingredient

Inactive Publication Date: 2013-08-22
THERALY PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]According to the present invention, by performing selective PEGylation, the yield of an exendin-4 analogue in which a cysteine (Cys) is introduced into #40 site of the C-terminal and is PEGylated with polyethylene glycol (PEG) or de

Problems solved by technology

However, traditional PEGylation employs nonspecific PEGylation methods that do not consider PEG reacting site, number of PEG bonds and biological activity.
However, such a nonspecific PEGylation method reduces treatment effects by bringing insufficient conformation by producing various branched type PEG-bonded isomers that have different physiochemical, biological and pharmacokinetic char

Method used

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  • Exendin-4 analogue pegylated with polyethylene glycol or derivative thereof, preparation method thereof, and pharmaceutical compostion for preventing or treating diabetes, containing same as active ingredient
  • Exendin-4 analogue pegylated with polyethylene glycol or derivative thereof, preparation method thereof, and pharmaceutical compostion for preventing or treating diabetes, containing same as active ingredient
  • Exendin-4 analogue pegylated with polyethylene glycol or derivative thereof, preparation method thereof, and pharmaceutical compostion for preventing or treating diabetes, containing same as active ingredient

Examples

Experimental program
Comparison scheme
Effect test

examples 1-5

C40 Site Specific PEG Bound Exendin-4 Production

[0051]To prepare C40 site specific PEG bound exendin-4, exendin-4-Cys in which cysteine is introduced into the C-terminal site (#40 site) was used (exendin-Cys, molecular weight: 4290.7, sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC), and maleimide activated monomethoxy PEG (mPEG-MAL, MW: 5, 20 kDa(Linear type), 20 kDa (Branch type), 23, 50 kDa (Trimer type)) was purchased from Nippon Oil and Fats, NOF, Tokyo, and used.

[0052]To prepare C-terminal #40 site specific PEG bound exendin-4, exendin-4-Cys and mPEG-MAL (MW: 5, 20 (linear type), 20 (branch type), 23, 50 kDa) were completely dissolved in a mole ratio of 1:2 in a 20 mM phosphate buffer saline (pH 7.5) and were reacted for two hours at room temperature (refer to FIG. 1). After the reaction, the reacted solution was separated by a reversed phase chromatography with Capcell-pak RP-18 column (250×10 mm, 5 μm, Shiseido, Japan) at a flow speed of 5.0 ml / min. The separation was mon...

experimental example 1

Analysis of RIN-m5F Cell Receptor Binding Affinity of PEG Bound Exendin-4 Analogue

[0061]The following experiment was performed to perform GLP-1 receptor (GLP-1R) affinity of PEG bound exendin-4 analogues of Example 1 (C40-PEG5K-Ex4), Comparative example 1a (Lys12-PEG5K-Ex4), Comparative example 1b (Lys27-PEG5K-Ex4) and Comparative example 2 (Nter-PEG5K-Ex4) prepared in Example 1, Comparative example 1 and 2.

[0062]Islet cells (RIN-m5F, ATCC, Manassas, Va.) expressing vast quantity of GLP-1 receptor (GLP-1R) were inoculated in 12-wells plates. It was washed twice with binding buffer (120 mM NaCl, 1.2 mM MgSO4, 13 mM sodium acetate, 5 mM KCl, 1.2 g / l Tris, 2 g / l bovine serum albumin, 1.8 g / l glucose, pH 7.6) after 48 hours and unmarked PEG bound exendin-4 analogue (final concentration range: 0.001-1000 nM) and exendin-4 marked with 30 pM concentration I-125 (9-39, PerkinElmer, Boston, Mass.) were treated simultaneously. Thorough washing was done with PBS including 1 mg / l bovine serum a...

experimental example 2

Evaluation of Low Blood Glucose Sustainability in Non-Fasting Type 2 Diabetic Mice

[0065]The following experiment was performed to evaluate low blood glucose sustainability of C40 site specific PEG bound exendin-4 composition according to the present invention in Type 2 diabetic mice.

[0066]Type 2 diabetic C57BL / 6 db / db mice (male, 4-5 weeks old, Central Lab. Animal Inc.) were used, and animals were exposed to light at 12 hours cycle and were grown after having stabilized two week by allowing free intake of foods and water. The experimental animals were managed following the guideline of National Institute of Health (NIH) and authorized by Institutional Animal Care and Use Committee of Sungkyunkwan University, and the experiment was performed humanely.

[0067]C40-PEG5K-Ex4 (linear), C40-PEG-Ex4 (linear), C40-PEG20K-Ex4 (branch), C40-PEG23K-Ex4 (trimer) and C40-PEG50K-Ex4 (trimer) prepared from the Example 1 to 5 and Lys27-PEG20K-Ex4 prepared in Comparative example 1 b were intraperitone...

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Abstract

The present disclosure relates to an exendin-4 analogue PEGylated with polyethylene glycol or a derivative thereof, a preparation method, and a pharmaceutical composition for prevention or treatment of diabetes containing the same as an active ingredient. According to the present invention, the yield of an exendin-4 analogue can be increased via the selective PEGylation by using exendin-4 in which a cysteine is introduced into #40 site of the C-terminal, and treatment effect of medications can be increased, so that the exendin-4 analogue can be usefully applied as a composition for prevention or treatment of diseases caused by insulin hypersecretion.

Description

TECHNICAL FIELD[0001]The present disclosure relates to an exendin-4 analogue PEGylated with polyethylene glycol or a derivative thereof, a preparation method thereof, and a pharmaceutical composition for prevention or treatment of diabetes containing the same as an active ingredient.BACKGROUND ART[0002]Among pharmaceutical technologies, PEGylation of peptides and proteins for the purpose of treatment is the most effective technology. PEGylation of peptides and proteins increases molecular weight thereof, protein degradation site defense and immunogenicity site defense, which consequently increases half-life of in vivo medications and reduce immunogenicity of peptides and proteins. Therefore, PEGylation technology has an effect of increasing treatment effect by solving problems of original medications, and due to such strength, serves an important role in increasing effects of PEGylated peptide and protein medication delivery system.[0003]Also, peptides and proteins increase treatmen...

Claims

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Application Information

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IPC IPC(8): A61K47/48
CPCA61K47/48215C07K14/57563A61K38/2278A61K9/0019A61K9/2018A61K9/2059A61K9/4858A61K9/4866A61K47/02A61K47/26A61K47/60A61P3/10A61K38/22A61K47/30A61K47/50
Inventor LEE, SUNG KWONKIM, WON BAELEE, SEULKIKIM, TAE HYUNG
Owner THERALY PHARMA
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