Insulin analogs and use thereof

Patent metadata

Field	Value
Publication number	US-10647753-B2
Application number	US-201815990495-A
Country	US
Kind code	B2
Filing date	May 25, 2018
Priority date	Aug 28, 2015
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

An insulin analog with an improved in vitro effect compared with native insulin, a pharmaceutical composition for treating diabetes containing the insulin analog as an active ingredient, and a method for treating diabetes using the insulin analog or the pharmaceutical composition are described. A nucleic acid encoding the insulin analog, an expression vector including the nucleic acid, a transformant introduced with the expression vector, and a method of producing the insulin analog from the transformant are also described.

First claim

The invention claimed is:

1. An insulin analog peptide comprising an A-chain of SEQ ID NO: 3 indicated in the following General Formula 1 and a B-chain of SEQ ID NO: 4 indicated in the following General Formula 2:
(General Formula 1)
Xaa1-Ile-Val-Glu-Xaa2-Cys-Cys-Thr-Ser-Ile-Cys-Xaa3-Leu-Xaa4-Gln-Xaa5-Glu-Asn-Xaa6-Cys-Xaa7  (SEQ ID NO: 3)

wherein
Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine;
Xaa2 is alanine, glutamic acid, glutamine, histidine, or asparagine;
Xaa3 is alanine, serine, glutamine, histidine, or asparagine;
Xaa4 is aspartic acid;
Xaa5 is alanine, leucine, tyrosine, histidine, or asparagine;
Xaa6 is alanine, tyrosine, serine, glutamic acid, histidine, or asparagine; and
Xaa7 is asparagine, glycine, histidine, or alanine; and
(General Formula 2)
Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa8-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Xaa9-Tyr-Xaa10-Xaa11-Lys-Thr  (SEQ ID NO: 4)

wherein
Xaa8 is tyrosine or is absent;
Xaa9 is phenylalanine or is absent;
Xaa10 is threonine; and
Xaa11 is proline or glutamic acid.

2. The insulin analog peptide of claim 1 , wherein, in the B-chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, and Xaa10 is threonine.

3. A pharmaceutical composition comprising the insulin analog peptide according to claim 1 and a pharmaceutically acceptable carrier.

4. A method of preparing the insulin analog peptide according to claim 1 , comprising:
a) preparing a recombinant expression vector comprising a nucleic acid encoding the insulin analog peptide according to claim 1 ;
b) transforming the recombinant expression vector into a host cell and obtaining a transformant therefrom;
c) culturing the transformant and expressing the insulin analog peptide; and
d) isolating and purifying the expressed insulin analog peptide.

5. The method of claim 4 , wherein the transformant is E. coli.

6. The method of claim 4 , wherein the isolating and purifying comprise:
d-1) obtaining the transformant cells from the culture and pulverizing the same;
d-2) recovering the expressed insulin analog peptide from the pulverized cell lysate followed by refolding the same;
d-3) purifying the refolded insulin analog peptide by cation exchange chromatography;
d-4) treating the purified insulin analog peptide with trypsin and carboxypeptidase B; and
d-5) sequentially purifying the treated insulin analog peptide by cation exchange chromatography and anion exchange chromatography.

Assignees

• Hanmi Pharm Ind Co Ltd

Inventors

• Kim Jin Young
• Oh Euh Lim
• Lee Jong Soo
• Lim Hyung Kyu
• Choi In Young
• Kwon Se Chang

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• C07K14/62 Primary

  Insulins · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title