WO2011063549A1

WO2011063549A1 - Long-acting exendin 4 analogues

Info

Publication number: WO2011063549A1
Application number: PCT/CN2009/001321
Authority: WO
Inventors: 吴晓琰; 龚铁军; 孙玉琨
Original assignee: Wu Xiaoyan; Gong Tiejun; Sun Yukun
Priority date: 2009-11-26
Filing date: 2009-11-26
Publication date: 2011-06-03
Also published as: CN102712690B; CN102712690A

Abstract

Provided is an Exendin 4 analogue peptide. Compared with wild-type Exendin 4, the peptide of the present invention is 7 amino acid residues shorter at the C-terminal end. The length of the peptide chain is similar to human GLP-1, and there is only one Lys20 amino acid residue in the structure of the peptide, such that acylation of fatty acid occurs specifically at theε-NH2 of the Lys20 to form a fatty acid acylated derivative. Also provided are pharmaceutical compositions comprising such peptide and methods for producing the peptide by chemical synthesis or recombinant DNA technologies.

Description

Long-acting Exendin 4 analogue

Technical field

The present invention relates to a polypeptide of an Exendin 4 analog which has a hypoglycemic effect and is useful for the treatment of type 2 diabetes. The invention also provides methods of producing these polypeptides by chemical synthesis and recombinant DNA production processes.

Background technique

The number of people with diabetes in the world has increased year by year. Now there are 50 million in China, 60 million in India, 20 million in the United States, and 6 million in Japan. The world has reached 220 million. There are two types of diabetes, one is insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes). Among them, type 2 diabetes accounts for more than 90% of diabetic patients.

Type 2 diabetic patients exhibit many characteristics, such as decreased β-cells, insufficient insulin secretion after meals, delayed secretion of insulin, increased fasting and postprandial blood glucose, elevated glycosylated hemoglobin HbAlc, and insulin resistance.

According to the US UKPDS multi-year follow-up study on type 2 diabetes, it is pointed out that none of the current 6 drugs can reverse the progression of diabetes in patients with type 2 diabetes, can not inhibit the progressive apoptosis of pancreatic β cells, and can not prevent the complications of diabetes. For example, the occurrence and development of coronary heart disease and renal failure change the prognosis of diabetic patients. Therefore, it is necessary to study new therapeutic drugs for type 2 diabetes.

Exendin 4 and Exendin 3 are two peptides secreted by the saliva of Gila monster, Heloderme Suspectum, which consists of 39 amino acid residues, chemically synthesized as an anti-type 2 diabetes drug. Approved by the US FDA in 2005, the trade name is Byetta, and it was listed in China in 2009.

Exendin 4 can promote the synthesis of proinsulin, promote insulin secretion, reduce fasting, postprandial blood glucose, and no longer continue to function when blood sugar is normal, so it is not easy to produce hypoglycemia, coma, shock, and high safety. It can reduce HbAl c , increase the number and volume of pancreatic β cells, increase the sensitivity of insulin receptors in type 2 diabetes patients, and inhibit the secretion of glucagon. Inject twice a day, 5-10ug each time. However, there are also shortcomings. Exendin 4 is derived from the giant lizard, and GLP-1 is a human endogenous substance. Clinical trials have reported that 52% of patients in Exendin 4 produce antibodies ( Diabetes (1997) 46, 433-439; Diabetes Care (2002) 25, 330-336; JAMA (2002) 287, 373-379; Diabetes (1995) 44. 1249-1258; Diatetes (2002) 51 2796-2803; Diabetes Care (2000) 23 64-69; Engl. J. Med (2002) 346, 393-463; Lancet (1998) 352, 837-853). In addition, Exendin 4 is a chemically synthesized product that is very expensive.

Summary of the invention

The object of the present invention is to address the current market demand for Exendin 4, which is very expensive, and the present specification provides a 32 amino acid polypeptide derivative of Exendin 4. Its peptide chain length is similar to that of human GLP-1, reducing its immunogenicity. The structure contains only one Lys20 residue, which allows the fatty acid acylation to specifically bind to Lys20 ε (epsi lon ) - ΝΗ 2 to form a fatty acid acylated derivative. In addition, the present invention utilizes genetic engineering technology to mass-produce the polypeptide, which can reduce the cost and provide a new therapeutic drug for a large number of type 2 diabetic patients. One aspect of the present invention provides a structural change, and still has hypoglycemic activity Exendin 4 Analog,

The Exendin 4 analog of the present invention has the following structural formula:

EW1 ( 1-32 )

His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-

Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-X3

EW2 (1-32)

His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-

Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Val-Gln-Gly-Gly-Pro-X3

Where: Xl=Phe or Tyr

X2=Met, Leu or He;

X3 = Arg, OH or NH2.

Preferably, XI is Tyr or Phe, X2 is Met or Leu, and more preferably, X3 is Arg.

Another aspect of the invention provides a pharmaceutical composition comprising the above Exendin 4 analogue, the composition comprising one or more compounds having the above formula and a pharmaceutically acceptable diluent or excipient, preferably The composition is in unit dosage form, such as tablets, pills, capsules (including sustained release or delayed release forms), powders, granules, elixirs, elixirs, syrups and emulsions, sterile injectable solutions or suspensions, aerosols Or liquid sprays, drops, injections, automatic injection devices or suppositories. For example, orally administered in the form of a tablet or capsule, the above active pharmaceutical ingredient may be combined with an oral non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Further, the present invention provides the use of the above compound and a pharmaceutical combination for the preparation of a therapeutic disease, preferably the disease is type 2 diabetes. A further aspect of the invention provides a genetic engineering method for the preparation of the above Exendi n 4 analogs and derivatives. The above compound in which X3 is Arg may be synthesized by a genetic engineering method or may be synthesized by a chemical method, but is preferably synthesized by a genetic engineering method. The above compounds in which X3 is 0H or NH2 can also be synthesized by a chemical synthesis method using a polypeptide synthesizer.

The invention changes the chemical structure of Exend in 4, shortens the length of the peptide chain from the C-terminus and changes the amino acid structure, and maintains its hypoglycemic activity, so that the peptide chain length of Exendi n 4 is similar to that of human GLP-1, and the immunogenicity is lowered. The peptide chain contains only one Lys20, which facilitates the site-direct acylation of fatty acids to form long-acting derivatives. At the same time, the need to adapt to the production of genetic engineering methods is suitable for mass production of genetic engineering, reducing costs, adapting to the needs of patients with type 2 diabetes, and providing a new therapeutic drug for patients with type 2 diabetes.

DRAWINGS

Figure 1 is a tandem map of Exendi n 4 analog gene fragments;

Figure 2 is a flow chart of the production of Exendi n 4 analog by genetic engineering;

Figure 3 is a measure of the hypoglycemic effect of EW1 and Exend i n4.

detailed description

Embodiment 1:

Production of EW 1 by genetic engineering

Material

EW 1

EcoRI Bgl II Arg Hi s Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Ser Gin Met Glu

^ _ ^ CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA Glu Glu Ala Val Lys Leu Phe l ie Glu Trp Leu Val Asn Gly Gly Pro Arg

GAA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT AAC GGC GGC CCG CGT GGATCC TAG ^ ~

BamH I Sal I

(1) synthesizing DNA fragments based on the EW1 amino acid sequence and the corresponding DNA (plus strand) sequence

5' AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG

AAGAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTAACGGCGGCCCGCGTGGATC CTAGTCGAC 3'

Synthetic DNA fragment

1) 5' AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG 3' 4) 5' CTAGGATCCACGCGGGCCGCCGTTAACCAGCC 3'

5) 5' GTGCCTTCGCCGTGACGAGATCTGG 3'

6) 5' tcgaCTAGGATCCACGCGGGCCGCCGTTAACCAGCC 3'

(2) The EW1 gene was synthesized by the conventional PCR method, and then digested with EcoR I /Sal I and cloned into the Lac promoter-containing plasmid. The DNA sequence was identical to the design.

TCGAC

(3) Referring to Figure 1, the EW1 gene was ligated in tandem to obtain a plasmid containing 8 copies of the gene. The complete DNA sequence, in accordance with the design, was transformed into E. col i JM109. Embodiment 2:

EW2

EcoR I Bgl II Asn Gly Arg His Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Ser Gin Met Glu ^ _ ^ ^ _ ^ AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA Glu Glu Ala Val Lys Leu Phe l ie Glu Trp Leu Val Gin Gly Gly Pro Arg

GAA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT GGATCC TAG ^ _ ,

BamH I Sa il synthesizes two DNA fragments using 1 1 of £ ₁ as a template

487f: 5' ccaatgAATTCCAGATCTAACGGCCGTCACGGCGAAG 3' 487r : 5' aattGTCGACTAGGATCCACGCGGGCCGCCCTGAACCAGCCAT 3'

PCR amplification was carried out, DNA sequences were determined, and EW2 genetically engineered bacteria were constructed.

gAAt TCC AGA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GA'

GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_

GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA

GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_

GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT GGA TCC TAGtcgac Example 3:

Preparation of EW1 and EW2

(A) Fermentation of genetically engineered bacteria: Ewl genetically engineered bacteria are inoculated into 300 ml X 2 bottles of LB medium, plus

Ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, 37 ° C, shaken overnight

(150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 g/ml, aeration of 20L/ _m in, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the concentration of the bacteria reached the neutral line of the log curve, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.

(B) Cell disruption: The collected cells were homogenized with pH 7.0 0 Tris buffer 20 mM, followed by lysozyme

Ig/L, incubate at 37 °C for 1 hour, freeze at 20 °C overnight, sonicate 3 times, each time for 1 minute, collect the precipitate by centrifugation, 2M Urea, crush and wash once, then wash with water and wash once.

(C) Separation and purification of inclusion bodies: Precipitate dissolved in 8M urea, molecular sieve Sephadex G-100 A2S0nm Detection, overnight dialysis, gradient elution with DEAE pH 8.0-9.0, protein electrophoresis to determine the target protein.

(D) Cleavage of the fusion protein: The dicarboxylic acid is dissolved, and 1 to 2% is added under pH 7.5. High-purity porcine trypsin 37Ό Stir 2~3 hours, HPLC trace. After the enzymatic hydrolysis reaction was completed, trypsin was removed by a peristaltic pump 30000 molecular weight ultrafiltration.

(E) Preparative HPLC purification.

(F) Freeze drying to obtain the product. Embodiment 4:

Preparation of myristic acid acylated EW2 derivative

(A) Take 10 g of EW2 fusion protein in 100 ml of water, add 50 ml of 2M hydroxylamine and adjust pH 9.0, add guanidine hydrochloride to a final concentration of 5 M, and incubate at 45 ° C overnight.

Weighing Marstic acid 1.14g, N-hydroxysuccinimide

(N-hydroxysuccinimide) 0.6g, Ν, Ν'-dicyclohexylcarbimide (DCCI) 1.03g dissolved in 50ml dioxane, stirred at room temperature, 4 hours later, filtered to remove urea derivatives 20 ml of the clear solution was added to (A), and Tetrameltyl-guanidine was added thereto. The reaction was carried out under vigorous stirring at room temperature for 4 hours, then adjusted to pH 3-4 with 2N HCl, centrifuged at 8000 rpm, and the precipitate was washed with ethanol. Two times, the fatty acid acylated Ew2 was dissolved in water, and the sodium hydrogencarbonate powder was adjusted to pH 7 to 8, 1 to 2% by weight. High purity porcine trypsin was stirred at 37 ° C for 2 to 3 hours and traced by HPLC. Preparative HPLC purification. Freeze drying to obtain the product. Embodiment 5:

Determination of hypoglycemic effect of EWi and EW2

C57BL/6 8-week-old mice, fasted overnight (only for drinking water)

Control group, EWi group, Exendin 4 group

4 rats in each group were injected intraperitoneally once, then 30 μg of 30% glucose solution was injected, glucose was injected every 2 hours, and venous blood was taken 5 μl after 20 minutes. Glucose oxidase test paper was used to measure fast blood glucose. 2, the results show that the hypoglycemic effect of EWi is comparable to Exendin 4.

Claims

Claim

1. Long-acting Exendin 4 analogue, its structural formula is:

EW1 ( 1 -32 )

His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-

Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-X3

Where: Xl=Phe or Tyr;

X2=Met or Leu or lie;

X3 = Arg or OH or NH2.

2. Long-acting Exendin 4 analogue, its structural formula is:

EW2 ( 1 -32)

His-Gly-Glu-Gly-Thr-Xl-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-

Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Val-Gln-Gly-Gly-Pro-X3

Where: Xl=Phe or Tyr;

X2=Met or Leu or l ie;

X3 = Arg or OH or NH2.

The Exendin 4 analog according to claim 1 or 2, wherein the peptide chain has a length of 32 amino acids.

The Exendi n 4 analog according to claim 1 or 2, wherein the X3 is 0H or -NH2.

The Exendin 4 analogue according to claim 1 or 2, wherein the ε (abbreviation of epsi lon)-NH2 of Lys ₂ Q is myristic acid or palmitic acid acylate.

A pharmaceutical composition comprising the Exendin 4 analogue according to claim 1 or 2, which further comprises a pharmaceutically acceptable diluent, excipient or carrier.

The pharmaceutical composition according to claim 6, wherein the carrier is selected from the group consisting of ethanol, glycerin and water.

The Exendin 4 analog according to claim 1 or 2, which is used for the preparation of a medicament for treating diabetes, wherein the diabetes is type 2 diabetes.

The pharmaceutical composition according to claim 6 or 7, which is used for the preparation of a medicament for treating diabetes, wherein the diabetes is type 2 diabetes.