GLP-1 ANALOGUE, ITS PREPARATION METHODS AND USE THEREOF

TECHNICAL FIELD

The present invention relates to the field of the medicaments associated with diabetes, specifically to a glucagon-like peptide-1 (GLP-1) analogue with a prolonged half-life of GLP-1 in vivo. The present invention also relates to a preparation method of the GLP-1 analogue and use of the GLP-1 analogue in the manufacture of a medicament for treating diabetes.

Background Art

The Glucagon-like peptide-1 (hereinafter referred to as GLP-1) involved in the present invention is a polypeptide consisting of 37 amino acids mainly secreted by small intestinal L cells, and the active forms of GLP-1 are GLP-1(7-37)OH and GLP-1 (7-36)NH2 (Mojsov S, J Clin Invest. 1987 February; 79(2): 616-9). GLP-1 can significantly reduce the blood glucose after meals in human, simulate the production of insulin, and meanwhile also play a role in reducing body weight without causing hypoglycemia (Drucker D J, Diabetes. 1998 February; 47(2): 159-69). Recent research also shows that GLP-1 has a pancreas regeneration effect (Drucker D J, 2003 December; 144(12): 5145-8). Moreover, GLP-1 is a fully humanized polypeptide, and thus possesses a great advantage in safely as a clinical drug. However, GLP-1(7-37) needs to be administered by injection for many times every day due to its serum half-life of only 3-5 minutes, and thus results in much inconvenience in the clinical use.

Recently, there are many researches using the GLP-1 analogue fusion protein technology to resolve the problem regarding the residence time of GLP-1 analogue in vivo (CN90101167.3, CN200710018734.2, CN200410054397.9, CN01820232.2, CN200380110152.7, CN200510039265.3, CN200610127237.1 and CN200910009642.7). However, the existing technologies are still far away from the ideal clinical goals, and generally fail to reach the clinical standard. Liraglutide, recently produced by Novo Norisk, is a GLP-1 analogue based on the modification of GLP-1 with palmitic acid, and has come into the market in America in 2009. However, Liraglutide also has the problem of a short half-life, and its dosage form still needs to be injected daily.

Therefore, there remains a need for a method of resolving the short half-life of GLP-1 in vivo.

DISCLOSURE OF THE INVENTION

Unless otherwise indicated, the “Fmoc strategy” as used herein refers to a synthetic method that amino acids with the amino terminal protected by Fmoc are condensed successively to synthesize a polypeptide in the presence of coupling reagent by using a polymer resin as the matrix of solid phase reaction. Specifically, please refer to Fmoc solid phase peptide synthesis: a practical approach, 2000, Oxford University Press.

In view of the defects that the clinically used GLP-1 analogues have short residence time in vivo and needs to be injected daily, one object of the present invention is to provide a GLP-1 analogue with longer half-life.

Another object of the present invention is to provide use of a GLP-1 analogue in the manufacture of a medicament for treating diabetes, and use of the GLP-1 analogue in the manufacture of a medicament for treating and/or preventing obesity.

Still another object of the present invention is to provide a pharmaceutical composition comprising the GLP-1 analogue described above as an active component, wherein the pharmaceutical composition further comprises one or more pharmaceutic ally acceptable auxiliary materials. Preferably, the pharmaceutical composition is an injection, further preferably is a lyophilized powder or a solution for injection.

The technical solutions for achieving the above objects are as follows:

In one aspect, the present invention provides a GLP-1 analogue having the following general formula A:

⁷HAEX₁₀TFTSX₁₅V SSYLEX₂₂QAAKEFIX₃₀W LX₃₃KGR G³⁷n₁X₁C n₂X₂ General Formula A,

wherein X₁₀is glycine or cysteine, X₁₅is aspartic acid or cysteine, X₂₂is glycine or cysteine, X₃₀is alanine or cysteine, X₃₃is valine or cysteine, and at least one of X₁₀, X₁₅, X₂₂, X₃₀and X₃₃is cysteine, n₁X₁means that the number of X₁is n₁, n₁=1-30, and X₁is glycine, alanine or valine; n₂X₂means that the number of X₂is n₂, n₂=0-30, and X₂is glycine, alanine or valine; and both of the two cysteines contained in the general formula A form disulfide bonds.

Preferably, the GLP-1 analogue has the following general formula I:

⁷HAEX₁₀T FTSX₁₅V SSYLE X₂₂QAAK EFIX₃₀W LX₃₃KGR G³⁷n₁X₁C n₂X₂ General Formula I,

wherein n₁=3-30, n₂=3-30.

The GLP-1 analogue having the general formula I such as:

SEQ ID NO 82:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR G³⁷

GGGGG GGGGG C GGGGG GGGGG;

SEQ ID NO 83:

⁷HAEGT FTSDV SSYLE CQAAK EFIAW LVKGR G³⁷

GGGGG C GGGGG GGGGG;

or

SEQ ID NO 84:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷

GGG C GGGGG GGGGGGGGGGGGGGG.

Preferably, the GLP-1 analogue has the following general formula II:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR G³⁷n₁X₁C n₂X₂ General Formula II,

n₁=1-20, n₂=0-25, and wherein X₁is glycine, alanine or valine, X₂is glycine, alanine or valine;

Preferably, n₁=3-20, n₂=3-25;

Further preferably, said n₁=5-15, n₂=3-9;

Further preferably, the GLP-1 analogue is:

SEQ ID NO 1:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG C GGG;

SEQ ID NO 2:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG GGGGGC GGG;

SEQ ID NO 3:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG GGGGGGGGGGC GGG;

SEQ ID NO 4:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG C GGGGG G;

SEQ ID NO 5:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG C GGGGG GGGG;

SEQ ID NO 6:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG GGGGGC GGGGG G;

SEQ ID NO 7:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG GGGGGC GGGGG GGGG;

SEQ ID NO 8:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG GGGGGGGGGGC GGGGG G;

SEQ ID NO 9:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷GGGGG GGGGGGGGGGC GGGGG GGGG;

SEQ ID NO 10:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA C AAA;

SEQ ID NO 11:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA AAAAAC AAA;

SEQ ID NO 12:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA AAAAAAAAAAC AAA;

SEQ ID NO 13:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA C AAAAA A;

SEQ ID NO 14:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA C AAAAA AAAA;

SEQ ID NO 15:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA AAAAAC AAAAA A;

SEQ ID NO 16:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA AAAAAC AAAAA AAAA;

SEQ ID NO 17:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA AAAAAAAAAAC AAAAA A;

SEQ ID NO 18:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷AAAAA AAAAAAAAAAC AAAAA AAAA;

SEQ ID NO 19:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV C VVV;

SEQ ID NO 20:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV VVVVVC VVV;

SEQ ID NO 21:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV VVVVVVVVVVC VVV;

SEQ ID NO 22:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV C VVVVV V;

SEQ ID NO 23:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV C VVVVV VVVV;

SEQ ID NO 24:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV VVVVVC VVVVV V;

SEQ ID NO 25:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV VVVVVC VVVVV VVVV;

SEQ ID NO 26:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV VVVVVVVVVVC VVVVV V;

or

SEQ ID NO 27:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR

G³⁷VVVVV VVVVVVVVVVC VVVVV VVVV.

Furthermore, the GLP-1 analogue having general formula A may also have the following general formula III:

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂ General Formula III,

wherein, n₁=1-10, n₂=0-25, X₁is glycine, alanine or valine, X₂is glycine, alanine or valine;

Preferably, n₁=3-10, n₂=3-25;

Or n₁=1-5, n₂=10-20;

More preferably, n₁=3-5, n₂=10-20.

Preferably, the GLP-1 analogue is:

SEQ ID NO 28:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷G

C GGGGG GGGGG;

SEQ ID NO 29:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷G

C GGGGG GGGGGGGGGG;

SEQ ID NO 30:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷G

C GGGGG GGGGGGGGGGGGGGGGGGGG;

SEQ ID NO 31:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷GG C GGGGG GGGGG;

SEQ ID NO 32:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷GG C GGGGG GGGGGGGGGG;

SEQ ID NO 33:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷GG C GGGGG GGGGGGGGGGGGGGG;

SEQ ID NO 34:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷GGGGG C GGGGG GGGGG;

SEQ ID NO 35:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷GGGGG C GGGGG GGGGGGGGGG;

SEQ ID NO 36:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷GGGGG C GGGGG GGGGGGGGGGGGGGG;

SEQ ID NO 37:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷A

C AAAAA AAAAA;

SEQ ID NO 38:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷A

C AAAAA AAAAAAAAAA;

SEQ ID NO 39:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷A

C AAAAA AAAAAAAAAAAAAAAAAAAA;

SEQ ID NO 40:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷AA C AAAAA AAAAA;

SEQ ID NO 41:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷AA C AAAAA AAAAAAAAAA;

SEQ ID NO 42:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷AA C AAAAA AAAAAAAAAAAAAAA;

SEQ ID NO 43:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷AAAAA C AAAAA AAAAA;

SEQ ID NO 44:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷AAAAA C AAAAA AAAAAAAAAA;

SEQ ID NO 45:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷AAAAA C AAAAA AAAAAAAAAAAAAAA;

SEQ ID NO 46:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷V

C VVVVV VVVVV;

SEQ ID NO 47:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷V

C VVVVV VVVVVVVVVV;

SEQ ID NO 48:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G³⁷V

C VVVVV VVVVVVVVVVVVVVVVVVVV;

SEQ ID NO 49:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷VV C VVVVV VVVVV;

SEQ ID NO 50:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷VV C VVVVV VVVVVVVVVV;

SEQ ID NO 51:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷VV C VVVVV VVVVVVVVVVVVVVV;

SEQ ID NO 52:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷VVVVV C VVVVV VVVVV;

SEQ ID NO 53:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷VVVVV C VVVVV VVVVVVVVVV;

or

SEQ ID NO 54:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LVKGR

G³⁷VVVVV C VVVVV VVVVVVVVVVVVVVV.

Furthermore, the GLP-1 analogue having general formula A may also have the following general formula IV:

⁷HAEGT FTSCV SSYLE GQAAK EFIAW LVKGR G³⁷n₁X₁C n₂X₂ General Formula IV,

wherein, n₁=1-10, n₂=0-30, X₁is glycine, alanine or valine, X₂is glycine, alanine or valine;

Preferably, n₁=3-10, n₂=3-30;

Or n₁=1-5, n₂=10-20;

More preferably, n₁=3-5, n₂=10-20.

Preferably, the GLP-1 analogue is:

SEQ ID NO 55:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷G

C GGGGG GGGGG;

SEQ ID NO 56:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷G

C GGGGG GGGGGGGGGG;

SEQ ID NO 57:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷G

C GGGGG GGGGGGGGGGGGGGG;

SEQ ID NO 58:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷GG C GGGGG GGGGG;

SEQ ID NO 59:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷GG C GGGGG GGGGGGGGGG;

SEQ ID NO 60:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷GG C GGGGG GGGGGGGGGGGGGGG;

SEQ ID NO 61:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷GGGGG C GGGGG GGGGG;

SEQ ID NO 62:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷GGGGG C GGGGG GGGGGGGGGG;

SEQ ID NO 63:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷GGGGG C GGGGG GGGGGGGGGGGGGGG;

SEQ ID NO 64:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷A

C AAAAA AAAAA;

SEQ ID NO 65:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷A

C AAAAA AAAAAAAAAA;

SEQ ID NO 66:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷A

C AAAAA AAAAAAAAAAAAAAA;

SEQ ID NO 67:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷AA C AAAAA AAAAA;

SEQ ID NO 68:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷AA C AAAAA AAAAAAAAAA;

SEQ ID NO 69:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷AA C AAAAA AAAAAAAAAAAAAAA;

SEQ ID NO 70:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷AAAAA C AAAAA AAAAA;

SEQ ID NO 71:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷AAAAA C AAAAA AAAAAAAAAA;

SEQ ID NO 72:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷AAAAA C AAAAA AAAAAAAAAAAAAAA;

SEQ ID NO 73:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷V

C VVVVV VVVVV;

SEQ ID NO 74:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷V

C VVVVV VVVVVVVVVV;

SEQ ID NO 75:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G³⁷V

C VVVVV VVVVVVVVVVVVVVV;

SEQ ID NO 76:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷VV C VVVVV VVVVV;

SEQ ID NO 77:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷VV C VVVVV VVVVVVVVVV;

SEQ ID NO 78:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷VV C VVVVV VVVVVVVVVVVVVVV;

SEQ ID NO 79:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷VVVVV C VVVVV VVVVV;

SEQ ID NO 80:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷VVVVV C VVVVV VVVVVVVVVV;

or

SEQ ID NO 81:

⁷HAEGT FTSDV SSYLE GQAAK EFICW LCKGR

G³⁷VVVVV C VVVVV VVVVVVVVVVVVVVV.

In another aspect, the present invention provides a preparation method of the GLP-1 analogue described above comprising solid phase polypeptide synthesis in accordance with Fmoc strategy.

In still another aspect, the present invention provides use of the GLP-1 analogue described above in the manufacture of a medicament for treating diabetes, obesity, diseases associated with diabetes and obesity associated with diabetes as well as for preventing obesity.

Moreover, the present invention provides a pharmaceutical composition comprising the GLP-1 analogue as claimed in any one of claims 1-8 and one or more pharmaceutic ally acceptable auxiliary materials.

Preferably, the pharmaceutical composition is an injection, further preferably is a lyophilized powder or a solution for injection.

Below is the detailed description of the present invention:

(1) GLP-1 Analogue

The general formula of the GLP-1 analogue described in the present invention is as follows:

⁷HAEX₁₀T FTSX₁₅V SSYLE X₂₂QAAK EFIX₃₀W LX₃₃KGR G³⁷n₁X₁Cn₂X₂ General formula A,

⁷HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G³⁷is a humanized GLP-1 sequence. The GLP-1 analogue of the above general formula A is an artificial sequence, in which the amino acid at position 10, 15, 22, 30 or 33 is modified by replacing the original amino acid with cysteine respectively. Furthermore, in the above general formula of amino acid, X₁may be Gly, Ala or Val; and X₂may be Gly, Ala or Val. At last, n₁and n₂in the general formula refer to that the number of the hydrophobic amino acid (Gly, Ala or Val) at C terminal of the polypeptide are n₁and n₂, n₁=1-30, n₂=0-30. In the above general formula of polypeptide, both of the two cysteines form disulfide bonds.

Preferably, the general formula of GLP-1 analogue is as follows:

⁷HAEX₁₀TFTSX₁₅V SSYLE X₂₂QAAK EFIX₃₀W LX₃₃KGR G³⁷n₁X₁C n₂X₂ General Formula I,

wherein n₁=3-30, n₂=3-30.

Preferably, the general formula of the mutated amino acid sequences having cysteines at positions 15, 30 and 33 are as follows:

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂ General Formula II,

n₁=1-20, n₂=0-25; preferably, n₁=3-20, n₂=3-25; wherein X₁is Gly, Ala or Val; X₂is Gly, Ala or Val.

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂ General Formula III,

n₁=1-10, n₂=0-25; preferably, n₁=3-10, n₂=3-25; wherein X₁is Gly, Ala or Val; X₂is Gly, Ala or Val.

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂ General Formula IV,

n₁=1-10, n₂=0-30; preferably, n₁=3-10, n₂=3-30; wherein X₁is Gly, Ala or Val; X₂is Gly, Ala or Val.

The above general formula II of the polypeptide may be further optimized as:

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂, n₁=5-15, n₂=3-9; wherein X₁is Gly, Ala or Val; X₂is Gly, Ala or Val.

The above general formula III of the polypeptide may be optimized as:

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂, n₁=1-5, n₂=10-20; preferably, n₁=3-5, n₂=10-20; wherein X₁is Gly, Ala or Val; X₂is Gly, Ala or Val.

The above general formula IV of the polypeptide may be optimized as:

⁷HAEGT FTSCV SSYLE GQAAK EFICW LVKGR G³⁷n₁X₁C n₂X₂, n₁=1-5, n₂=10-20; preferably, n₁=3-5, n₂=10-20; wherein X₁is Gly, Ala or Val; X₂is Gly, Ala or Val.

(2) Pharmaceutical Composition of the Present Invention

The GLP-1 analogue of the present invention can be prepared into a pharmaceutical composition in combination with one or more pharmaceutically acceptable auxiliary materials including water soluble filler, pH regulator, stabilizer, water for injection, osmotic pressure regulator and so on.

The water soluble filler of the present invention is one or more selected from the group consisting of mannitol, low molecular weight dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose, and so on.

The pH regulator is one or more selected from the group consisting of non-volatile acids, such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid and etc.; and physiologically acceptable organic or inorganic acids, bases and/or salts and so on, such as potassium hydroxide, sodium hydroxide or potassium hydroxide or ammonium hydroxide, sodium carbonate or potassium carbonate or ammonium carbonate, sodium bicarbonate or potassium bicarbonate or ammonium bicarbonate, and the like.

The stabilizer is one or more selected from the group consisting of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium dodecyl sulfate or trihydroxymethyl aminomethane and so on. The sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000 and trihydroxymethyl aminomethane are preferred.

The osmotic pressure regulator is one or the combination of sodium chloride and potassium chloride.

(3) Preparation Method of Injection

The pharmaceutical composition of the present invention can be administered by intramuscular, intravenous, or subcutaneous injection, and the preferable dosage form is a lyophilized powder or a solution for injection.

The preparation method of the freeze-drying injection

To an appropriate amount of GLP-1 analogue solution, water soluble filler, stabilizer, osmotic pressure regulator and the like are added, and an appropriate amount of water for injection is added. The pH value is adjusted to 4-8 so as to dissolve the materials therein. The resulting solution is diluted to a proper concentration by adding water. 0.1-0.5% of active carbon is added to the solution, and then removed after the solution is stirred for at 0-10° C. for 10-20 minutes. The resulting solution is filtered with microfiltration membrane to remove bacteria. The filtrate is subpackaged, and then fabricated in accordance with freeze-drying method as a white, loose and blocky substance, which is sealed to obtain the freeze-drying injection. Each specification contains the GLP-1 analogue between 5μg and 1 mg.

Preparation Method of the Solution for Injection

To an appropriate amount of GLP-1 analogue solution or freeze-dried powder, water soluble filler, stabilizer, osmotic pressure regulator and the like are added, and an appropriate amount of water for injection is added. The pH value is adjusted to 4-8 so as to dissolve the materials therein. The resulting solution is diluted to a proper concentration by adding water. 0.1-0.5% of active carbon is added the solution, and then removed after the solution is stirred at 0-10° C. for 10-20 minutes. The resulting solution is filtered with micro filtration membrane to remove bacteria. The filtrate is subpackaged, and sealed to obtain the solution for injection. Each specification contains the GLP-1 analogue between 5 μg and 1 mg.

(4) Use of Pharmaceutical Composition

The GLP-1 analogue of the present invention can be used in the manufacture of a medicament for treating diabetes. Specifically, the composition of the present invention can be administered in the form of intravenous, intramuscular, or subcutaneous injection. Although the dosage is varied depending on the subject to be treated, mode of administration, symptoms and other factors, the GLP-1 analogue of the present invention is effective in a wide range of doses. In the treatment of the adults, the dosage range is between 5 μg/person and 1 mg/person, administered once daily or every several days. The actual dosage should be determined by a physician according to related conditions including the physical state of the patient to be treated, administration route, age, weight, individual response to the drug, severity of the patients' symptoms and the like. Therefore, the above dosage range does not limit the scope of the present invention in any way.

The GLP-1 analogue of the present invention has overcome the problem of the short half-life of GLP-1. The half-life of the GLP-1 analogue provided can reach above 15-75 hours in vivo, which is significantly longer than that of GLP-1 administered alone (with a half-life of only 3-5 minutes), thereby are greatly favorable for the clinical spreading and application of the GLP-1 analogue of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The examples of the present invention are illustrated in detail below with reference to the drawings, in which:

FIG. 1 is the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 1-27) in Example 2; wherein FIG. 1-A represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 1-9), in which ten data bars corresponding to each of the time points represent SEQ 1-9 and the control successively from left to right; FIG. 1-B represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 10-18), in which ten data bars corresponding to each of the time points represent SEQ 10-18 and the control successively from left to right; FIG. 1-C represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 19-27), in which ten data bars corresponding to each of the time points represent SEQ 19-27 and the control successively from left to right;

FIG. 2 is the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 28-54) in Example 3; wherein FIG. 2-A represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 28-36), in which ten data bars corresponding to each of the time points represent SEQ28-36 and the control successively from left to right; FIG. 2-B represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 37-45), in which ten data bars corresponding to each of the time points represent SEQ37-45 and the control successively from left to right; FIG. 2-C represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 46-54), in which ten data bars corresponding to each of the time points represent SEQ46-54 and the control successively from left to right;

FIG. 3 is the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 55-81) in Example 4; FIG. 3-A represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 55-64), in which eleven bars corresponding to each of the time points represent SEQ55-64 and the control successively from left to right; FIG. 3-B represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 65-74), in which eleven data bars corresponding to each of the time points represent SEQ65-74 and the control successively from left to right; FIG. 3-C represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 75-81), in which eight data bars corresponding to each of the time points represent SEQ75-81 and the control successively from left to right;

FIG. 4 represents the blood glucose reducing test of the GLP-1 analogue (SEQ ID NO 82-84) in Example 5, in which four bars corresponding to each of the time points represent SEQ82-84 and the control successively;

FIG. 5 is a graph describing the stability of the GLP-1 analogue in the human serum in Example 6;

FIG. 6 is a graph describing the stability of the GLP-1 analogue in the human serum in Example 7;

FIG. 7 is a graph describing the stability of the GLP-1 analogue in the human serum in Example 8.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

The present invention is now further described in detail in combination with the specific embodiments. The examples given are only for illustrating the present invention and not limiting the scope of the present invention.

In the following examples, various processes and methods which are not described in detail are conventional methods known in the art.

Example 1
Solid-Phase Synthesis of Polypeptide

Using the method of solid phase polypeptide synthesis in accordance with Fmoc strategy, the synthesis of the polypeptides of the present invention is performed using the CS 336X type apparatus produced by CSBio Company. The method of synthesis is performed in accordance with the manufacturer's equipment instructions.

The obtained polypeptides are purified on a HPLC C18 semi-preparative column, using acetonitrile as the mobile phase. The freeze dried powder is obtained through desalination and lyophilization. All the polypeptides obtained in the present invention contain disulfide bonds, and ammonium bicarbonate or other reducing agent is used to form the disulfide bonds in the polypeptides.

Example 2
Related Blood Glucose Reducing Function of the GLP-1 Analogue (General Formula II)