Human insulin analogue and acylated derivative thereof

Patent metadata

Field	Value
Publication number	US-9458219-B2
Application number	US-201214363328-A
Country	US
Kind code	B2
Filing date	Nov 22, 2012
Priority date	Dec 15, 2011
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

The present invention provides a human insulin analog, an acylated derivative thereof and a physiologically acceptable salt. The present invention further provides a preparation method for the insulin analog and an application of the insulin analog as a therapeutic agent, and particularly as a diabetes mellitus therapeutic agent.

First claim

The invention claimed is:

1. A human insulin analogue or a physiologically acceptable salt thereof having an A chain and a B chain as follows:

wherein:
A 0 is omitted;
A 21 is N or G;
B 3 is N;
B 27 is T;
B 28 is D;
B 29 is K;
B 30 is E;
B 31 and B 32 are omitted;
the ε-amino group of the lysine residue at B29 is optionally acylated; and
optionally, the α-amino group at the N-terminus of the A chain or B chain is acylated.

2. The human insulin analogue or the physiologically acceptable salt thereof according to claim 1 , wherein the sequences of the A chain and B chain are selected from the group consisting of:

A chain: 
SEQ ID NO: 1

GIVEQCCTSICSLYQLENYCN

B chain: 
SEQ ID NO: 10

FVNQHLCGSHLVEALYLVCGERGFFYTDKE

and

A chain: 
SEQ ID NO: 2

GIVEQCCTSICSLYQLENYCG

B chain: 
SEQ ID NO: 10.

FVNQHLCGSHLVEALYLVCGERGFFYTDKE

3. The human insulin analogue or the physiologically acceptable salt thereof according to claim 1 , wherein the human insulin analogue is PEGylated.

4. The human insulin analogue or the physiologically acceptable salt thereof according to claim 1 , wherein the human insulin analogue is modified by PEG, and the molecular weight of the PEG molecule is 5-100 kD; and the PEG molecule is a branched-chain or straight-chain type.

5. An expressed precursor used for the preparation of the human insulin analogue or the physiologically acceptable salt thereof according to claim 1 , having the following formula (I):
B-R1-A  (I)

wherein:
R1 is a peptide fragment having 0 to 5 amino acid residues, wherein the peptide fragment consists of alanine (A), lysine (K) and arginine (R); and
A and B correspond to the A chain and B chain of the human insulin analogue, respectively, wherein the expressed precursor is selected from the group consisting of: SEQ ID NO: 19: FVNQHLCGSHLVEALYLVCGERGFFYTDKEKRGIVEQCCTSICSLYQLENYCN, and SEQ ID NO:77: FVNQHLQGSHLVEALYLVCGERGFFYTDKEKRGIVEQCCTSIQSLYQLENYCG.

6. A DNA encoding the expressed precursor according to claim 5 .

7. An expression vector comprising the DNA according to claim 6 .

8. A host cell transformed with the expression vector according to claim 7 .

9. The host cell according to claim 8 , wherein the host cell is a bacterium.

10. The host cell according to claim 8 , wherein the host cell is yeast.

11. An insulin derivative, comprising an acylated group connected to the α-amino group at the N-terminus of the A chain or B chain, or to the 8-amino group of a lysine residue at B29 of the human insulin analogue or the physiologically acceptable salt thereof of claim 1 , the insulin derivative has the following formula:
S-W-X—Y—Z

wherein S is the human insulin analogue according to claim 1 ;
-W-X—Y—Z is an acylated group of the human insulin analogue, wherein, W is selected from the group consisting of:
(i) a group having a di-acyl structure of —OC(CH2)mCO—, wherein m is an integer of 2 to 10, an amide bond is formed between one carboxyl group on the structure and the α-amino group of the N-terminal amino acid residue of the A-chain or B-chain, or the ε-amino group of a Lys residue existing in the B-chain of the human insulin analogue; and
(ii) an α-amino acid residue with a carboxyl group on the side chain or a peptide having 2 to 4 α-amino acids with a carboxyl group on the side chain, wherein an amide bond is formed between the residue or the peptide and the α-amino group at the N-terminus of the A-chain or B-chain, or the ε-amino group at a Lys residue existing in the B-chain of the human insulin analogue;

X is selected from the group consisting of:
(i) —CO—; and
(ii) a diamino compound comprising a carboxyl group, wherein an amide bond is formed between one of the α-amino groups of the diamino compound and the carboxyl group of W;
provided that:
a) when W is an α-amino acid residue or a peptide having 2 to 4 α-amino acids, the amide bond is formed between the amino group of W and the —CO— of X; or
b) when W is a group having a di-acyl structure, the X group is linked to the di-acyl structure via one of its amino groups;

Y is selected from the group consisting of:
(i) -A(CH 2 ) m —, wherein m is an integer of 6 to 32, and A is absent or is CO—;
(ii) a bivalent hydrocarbon chain comprising an acyl group, which comprises 1, 2 or 3 —CH═CH— groups and a number of —CH2- groups sufficient to result in a total of 10-32 carbon atoms in the chain; and
(iii) a bivalent hydrocarbon chain having the formula of -B(CH 2 ) v C 6 H 4 (CH 2 ) w —, wherein B is absent or is CO—, each of v and w is an integer or one of them is 0, making v and w in the range of 6 to 30;
provided that:
a) when X is CO—, A or B is absent; or
b) when X is a diamino compound, A or B is CO—;

Z is selected from the group consisting of —OH, —NH 2 , —COOH, —SO 3 H, and —PO 3 H.

12. The insulin derivative according to claim 11 , wherein the acylated group -W-X—Y—Z is connected to the 8-amino group of a lysine residue at B29 of the human insulin analogue.

13. The insulin derivative according to claim 11 , wherein the acylated group -W-X—Y—Z is connected to the α-amino group at the N-terminus of the A chain or B chain of the human insulin analogue.

14. The insulin derivative according to claim 11 , wherein S is a human insulin analogue is consisting of: A chain: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and B chain: FVNQHLCGSHLVEALYLVCGERGFFYTDKE (SEQ ID NO: 10).

15. The insulin derivative according to claim 11 , wherein the acylated group -W-X—Y—Z is selected from the group consisting of:
N α —(HOOC(CH 2 ) 14 CO)-γ-Glu;
N α —(HO(CH 2 ) 15 CO)-γ-Glu;

Assignees

• Shanghai hengrui pharmaceutical co ltd
• Jiangsu Hengrui Medicine Co

Inventors

• Sun Piaoyang
• Zhang Lianshan
• Liu Jiajian
• Yuan Jijun
• Fang Chunqian
• Sun Changan
• Yuan Hengli
• Wang Yali

Classifications

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• A61P5/50

  for increasing or potentiating the activity of insulin · CPC title

• A61K47/50

  the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates · CPC title

• 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

• C07K14/62 Primary

  Insulins · CPC title