CN100535006C

CN100535006C - Analog of GLP-1

Info

Publication number: CN100535006C
Application number: CNB2004100542995A
Authority: CN
Inventors: 孙玉琨; 张培璋; 周波
Original assignee: Huayi Bio-Technology Co Ltd Shanghai
Current assignee: Shanghai Benemae Pharmaceutical Corp
Priority date: 2004-09-06
Filing date: 2004-09-06
Publication date: 2009-09-02
Anticipated expiration: 2024-09-06
Also published as: CN1746188A

Abstract

The present invention provides a GLP-1 analog, which is a structure change polypeptide of GLP-1. The polypeptide has the action of reducing blood sugar, and the performance of the polypeptide is more high efficiency and stable than a parent body GLP-1. The present invention can be used for treating II type diabetes. In addition, the present invention also provides a method for preparing the polypeptide.

Description

The GLP-1 analogue

Invention field

The present invention relates to the GLP-1 analogue, the present invention relates to GLP-1 particularly and change the structure polypeptide, this polypeptide has the lowering blood glucose effect, and character is more efficient than parent GLP-1, stable, can be used for treating type ii diabetes.The present invention also provides the method for producing these polypeptide by chemosynthesis and recombinant DNA production technique.

Background technology

Countries in the world diabetic subject's number increases year by year, and is state-owned 4,000 ten thousand in now, India 6,000 ten thousand, the U.S. 1,800 ten thousand, Japan 6,000,000, the diabetic subject is divided into two kinds, and the one, insulin-dependent diabetes mellitus (type i diabetes) and non insulin dependent diabetes (II diabetes).Wherein, type ii diabetes accounts for more than 90% of diabetic subject.

The type ii diabetes patient has showed many features, as insulin secretion quantity not sufficient after the meal, and insulin secretion time lag, hyperglycemia etc.Fat type ii diabetes patient periphery cell Regular Insulin receptor sensitivity reduces, thereby produces the blood sugar height, the also high situation of insulin level in the blood, and glycolated hemoglobin, HbAlc is (normally being 4～6%) more than 8%.Can produce diabetic complication with that, as heart trouble and renal failure etc.

Nowadays, control of diabetes has 6 class medicines:

● insulin secretion accelerating class: sulfonylurea, melitioneds class

● the short secretion of non-insulin medicine: Regular Insulin, alpha-glucosidase inhibitor, biguanides and Thioagolinediones.

According to Britain UKPDS thousands of type ii diabetes patients' follow-up study in 6 years report is pointed out that above-mentioned 6 class medicines are all powerless to the type ii diabetes patient, can not contain the continuous progressive deterioration of pancreas beta cell, the HbAlc level can not be reduced, the complication of diabetes such as heart trouble, renal failure can not be stoped.Therefore need the new type ii diabetes medicine of research.

Found a kind of intestinal hormones GLP-1 (7-36)-NH in 1985 ₂, it is a peptide species of small intestine L-emiocytosis, can promote insulin secretion, lowering blood glucose.External source gives glucose level normalizing (the Diabetes Care (1992) that GLP-1 can make the type ii diabetes patient 15, 270-276; Lancet (2002) 359, 824-830; Endoer.Rev (1996) 16, 390-410; Diabeto1ogia (1985) 28, 565-573).

GLP-1 has following function:

● can promote insulinogenic synthetic, insulin secretion accelerating, lowering blood glucose

● improve the beta cell function, increase the beta cell amount

● increase peripheral cell Regular Insulin receptor sensitivity

● reduce HbAlc

(Diabetes(2002) 51，1443-1452；Diabetologia(2002) 45，1263-73；Diabetes 525-529(2001)；Diabetes(2001) 50，725；Diabetes(2003) 52，365-371；Recent Prog.Hormne Res.(2001) 56，377-399；Disbetologia(1996) 39，1546-1553；Am.J.Physicl Endocrinol.Metab.(2001) 281，E242-247；J.Clin.Endocrinology and Metabolism(2003) 88，3082-3089；Diabetes(2002)Diabetes Care(2002) 22 403-408(United KingdomProspective Diabetes Study)(UKPDS Reports 57)；N.Engl.J.Med，UKPDS Reports；Diabetes Care(2000) 23 64-69；Lancet(2002) 152072-2077；Diabetes S 1：2796-2803；U.S.patent 477967，478017，478425；Diabetes Care(1999) 22 403-408；J.Clin.Endocrinology andMetabolism(2003) 88 3082-3089；Diabetes(1995)44，1295；RestoregenReport July 5，2001)。

In addition, also there is acceptor in GLP-1 in cranial nerve cell, GLP-1 enters in the brain and receptors bind GLP-1 by the brain barrier, effective in cure to senile dementia (Alzheimer ' s disease), (Amyloid-β-peptide) content reduces to make short dextrin shape beta polypeptide, and can modify its precursor-dextrin amyloid protein precursor (Amyloid Protein Precursor, APP), GLP-1 particularly, Exendin4 can not oxidized dose of neuroprotective archeocytes or L-glutamic acid effect and apoptosis (J.Pharmaceology ﹠amp; Experimental Therapeutics (2002) 302881-888; J.Nearosa Res. (2003) 72603-612; J.Biol.Chem. (2003) 278471-478; J.Alzheimer ' s Disease (2002) 487-496).GLP-1 is to (the J.Nutr. (2003) that also plays an important role that loses weight 1332326-2330; Inter.J.Obesity (2001) 25781-792; J.Chin.Invest. (1998) 101515-520; Eur.J.Pharmacology (2002) 449269-279).

In blood, GLP-1 is subjected to the effect excision N-of pepx (DPPIV) to hold dipeptides and rapid the decomposition, and therefore a lot of people study and can resist its change structure (U.S. Patent No. 5118,666 that pepx is degraded; PCT/US 87101005; WO 87/06941; WO 90/11296; PCT/US 89101121).

1, modifies the C-end of GLP-1 (7-36) and the amino-acid residue in the molecule

GLP-1 (7-36) is that its structure is by the polypeptide of people's small intestine L-emiocytosis: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Le u-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Ly s-Gly-Arg.

Because GLP-1 is subjected to pepx (DPPIV) effect to be degraded to GLP-1 (9-36) rapidly in blood, loses blood sugar reducing function, therefore in order to prolong the transformation period (t of GLP-1 _1/2), many-sided research is carried out in a lot of in the world laboratories, and a kind of is inhibitor such as the NN2211 of research DPPIV, and the amino-acid residue in the displacement GLP-1 molecule that has is as Gly ⁸-GLP-1 (7-36), the degraded that can resist DDPPIV, but insulin secretion accelerating and the hypoglycemic activity of Gly8-GLP-1 (7-36) greatly reduce the AA with GLP-1 (7-37) that has than parent GLP-1 ⁸Be replaced as D-type amino acid, as, Ala ⁸-GLP-1, or AA ⁹-GLP-1, promoting insulin secretion is all well below parent GLP-1 (7-36) as a result.In addition, also have C-end to add 6 Lys residues, i.e. GLP-1 (7-36)-(Lys) with GLP-1 ₆Also have with Gly ⁸The C-of-GLP-1 (7-36) end adds the Ex4 (31-33) of the C-end of Exendin4, Ex4 (31-36) and Ex4 (31-39), and wherein, GLP-1 holds 9 amino-acid residues adding Ex4C, Gly ⁸-GLP-1 (7-36) Ex4-(31-39) returns to the Degradation that parent GLP-1 activity level can be resisted DPPIV.GLP-1 (7-36) becomes 39 identical (J.Chin.Endocrinol.Melab. (1995) with Exendin4 peptide chain length by 30 amino-acid residues like this 80952-957; Trend in Pharmacilogical Sci. (2003) 24377-383; RegulatoryPeptides (2003) 114153-158; WO 03/011892 A2, (2003); WO01/04156 (2001)).

Modify the GLP-1 side chain radical because of prolonging its transformation period t _1/2(J.Bilh.Chem. also succeeds 27221201-21206 (97); Inter.J.Peptide Protein Res. (1992) 40333-343; Endocrinology (1989) 1253109-3114; Regulatory Peptides (1999) 7993-102, (2003) 14151-158; Bilchemistry (2001) 402860-2869; US Patent (1992) 5118666; WO 03/011892, (PCT/US02/24141); Trend in PharmacologicalSci.24 (2003) 378-383; J.Med.Chem. (2000) 431664-1669; Diabetologia (1998) 41271-278).

2, modify the N-end of GLP-1 (7-36)

GLP-1 (7-37), add 1 or several amino acid residue outside the His of the N-end of GLP-1 (7-36) or remove two amino-acid residues, then there is not promoting insulin secretion, as GLP-1 (1-37), GLP-1 (6-37), GLP-1 (8-37) do not have promoting insulin secretion (Endocribology (1989) 1283109-3114; Regulatory Peptide (1999) 7993-102; Biochemistry (2001) 402860-69).

Up to now, the molecular modification result to GLP-1 (7-36), comprehensively list in the table:

Yet the molecular structure of above-mentioned GLP-1 changes the GLP-1 blood sugar reducing function that does not all have acquisition to significantly improve.The present invention obtains the GLP-1 analogue by GLP-1 being changed structure research, and these analogues show hypoglycemic activity and the stability that greatly improves than parent, are suitable for treating type ii diabetes.In addition, the GLP-1 receptor binding capacity that the present invention does not adopt external (in vitro), the formation ability of cAMP and the method for short insulinoma cell excreting insulin, and directly utilizing (in vivo) blood sugar decreasing effect in the body to estimate structure and the active relation of GLP-1, this direct mensuration hypoglycemic activity has direct directive significance to the clinical application of type ii diabetes.

Summary of the invention

The invention provides the GLP-1 analogue, this GLP-1 analogue has the general formula of formula I:

1 10 20 30

HX ₁EGTFTSDVSSYLEGQAAKEFLAWLVKGPPSX ₂(formula I)

X ₁=Ala or Gly,

X ₂=Arg-OH, OH or NH ₂

The present invention also provides the method for preparing above-mentioned GLP-1 analogue in addition, and making to vast type ii diabetes patient provides the GLP-1 medicine to become possibility in a large number.

Brief description of drawings

Fig. 1 is a synthetic GLP-1 analogue gene segment sequence chart;

Fig. 2 is the schema that gene engineering research is produced the GLP-1 analogue;

Fig. 3 is the structure synoptic diagram that contains the plasmid of multiple copied GLP-1 gene;

Fig. 4 is with the synthetic GLP-1 (7-35) of solid-phase synthesis-PPSR and G ⁸The synoptic diagram of-GLP-1 (7-35)-PPSR

Fig. 5 A-C is the blood sugar decreasing effect figure of GLP-1 analogue of the present invention;

Fig. 6 is the insulin secretion accelerating design sketch of GLP-1 analogue of the present invention.

Detailed Description Of The Invention

One aspect of the present invention provides structure to change, and the GLP-1 analog of the higher more stable formula (I) of hypoglycemic activity. Preferably, X₁Be Ala or Gly, X₂Be Arg-OH. At this moment, the structural formula of GLP-1 analog is that His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Leu-Ala-Trp-Leu-Val-Lys-Gly-Pro-Pro-Ser-Arg-OH or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Leu-Ala-Tr p-Leu-Val-Lys-Gly-Pro-Pro-Ser-Arg-OH are respectively referred to as GLP-1 (7-35)-PPSR and Gly⁸-GLP-1(7-35)-PPSR。

In like manner, work as X₁Be Ala, X₂Be OH or NH₂The time respectively referred to as GLP-1 (7-35)-PPS-OH and GLP-1 (7-35)-PPS-NH₂ Work as X₁Be Gly, X₂Be OH or NH₂The time respectively referred to as Gly⁸-GLP-1 (7-35)-PPS-OH and Gly⁸-GLP-1(7-35)-PPS-NH ₂。

Another aspect of the present invention provides the composition that contains above-mentioned GLP-1 analog, said composition contains compound and a kind of pharmaceutically acceptable diluent or the excipient of one or more formulas I, preferably said composition is unit dosage form, such as tablet, pill, capsule (comprising sustained release or delayed release form), pulvis, particle, elixir, tincture, syrup and emulsion, injection solution or the suspension of sterilization, aerosol or liquid spray, drops, injection, automated injection device or suppository. For example, with tablet or capsule ground form oral administration, above-mentioned active medicine component can with a kind of oral acceptable inert carrier of nontoxic pharmacy, such as ethanol, glycerine, water and analog combination thereof. In addition, the present invention also provides the purposes in preparation treatment disease of above-claimed cpd and pharmaceutical composition, and preferably this disease is diabetes.

Another aspect of the present invention provides the method for preparing above-mentioned GLP-1 analog, comprises chemical synthesis process and gene engineering method. For X₂＝NH ₂Analog, can synthesize with chemical method. For X₂The analog of=Arg-OH or OH can both can be used chemical synthesis production, also can use gene engineering method production, preferably used gene engineering method. Chemical method wherein is solid-phase synthesis preferably. Certainly can also adopt other conventional method well known in the art to synthesize.

The genetic engineering process for preparing of polypeptide is compared with albumen and is acquired a certain degree of difficulty, when the polypeptide of the moderate-length of the using gene engineering technique preparation molecular weight resemble GLP-1, for the polypeptide than small-molecular weight of avoiding cell to express digests, usual way is that the gene of carrier protein (carrier protein) that its gene and another molecular weight is larger is linked together, again with the formal representation of fusion out. In addition, fusion will carry out cracking, separation etc. through suitable method just can obtain desired polypeptides, is subject to the restriction of many technical elements. GLP-1 content is less in the fusion that general gene engineering method obtains, and in complicated protein mixture, further separation and purification GLP-1 difficulty is very large, is worth without production and application after the cracking.

By analysis and research, the gene that the present invention expresses the GLP-1 analog with n (n=2～32) is together in series and carries out plasmid structure (Fig. 3). Use the genetic engineering bacterium of the thinking structure of this uniqueness, the fusion of its expression is comprised of n GLP-1 analog, through with suitable method cracking, just can produce n GLP-1 analog again. So not only improved the expression of GLP-1 analog, also greatly reduced the step of Downstream processing, thereby established good technical foundation for low-cost production GLP-1 analog in enormous quantities. According to the amino acid sequence of GLP-1 analog, select synthetic four the corresponding dna fragmentations of the maximum codon of Escherichia coli frequency of utilization.

Embodiment 1: produce GLP-1 (7-35)-PPSR with gene engineering method

Material

Cloning vector contains the lac promoter plasmid; Host bacterium JM103:Promega company; T4 polynucleotide kinase test kit: Promega company; T4 ligase enzyme test kit: Promega company; Restriction enzyme EcoR I, Sal I, Bgl II, BamH I test kit: Promega company; Molecular weight standard λ DNA, φ * 174DNA:Promega company; Lower molecular weight standard protein: Shanghai Si Ji biological products company limited; ATP (Adenosine-5 '-triphosphate, adenosine-5 '-triphosphoric acid): Boehringer Mannheim company; Acrylic amine: E.Merck; Methylene diacrylamide: Fluka; Ethidium bromide (Ethidum bromid): Serva; Tris:A.R Shanghai Si Ji biological products company limited (import packing); SDS:A.R Shanghai Si Ji biological products company limited (import packing).

LB nutrient solution: tryptone (Tryptone): Oxoid, yeast extract powder: Oxoid, sodium-chlor (A.R): chemical reagent station, Chinese Medicine group Shanghai.

Other chemical reagent: be the AR level, chemical reagent station, Chinese Medicine group Shanghai provides.

The connection of gene fragment

1)5’-AAT TCC AGA TCT ATG CGT CAC GCG GAA GGT ACC TTCACC AGC GAT GTG GTG AGC AGC TAT CTG-3’

2)5’-GAA GGT CAG GCG GCG AAA GAA TTT ATC GCG TGG CTGGTG AAA GGT CCG CCG AGC CGT TCC TAG G-3’

3)5’-TGGAC CTA GGA TCC ACG GCT CGG CGG ACC TTT CACCAG CCA CGC GATAAATTC TTT CGC CGC CTG-3’

4)5’-ACC TTC CAG ATA GCT GCT CAC ATC GCT GGT GAA GGTACC TTC CGC GTG ACG CATAGATCT GG-3’

(content is A with above four gene fragments _260nm=3 (0.D.)) respectively add distilled water 30 μ l.Get each 2 μ l of positive minus-strand dna fragment and be mixed in (i.e. (1)+(3) and (2)+(4)) in two plastics tubings respectively, and add following reagent and carry out 5 ' and hold phosphorylation reaction:

10 * polynucleotide kinase damping fluid

1μl

ATP(0.1mol/L)	1μl
ATP(0.1mol/L)	1μl	Distilled water	3μl
The T4 polynucleotide kinase	1μl	Distilled water	3μl

Mix the back in 37 ℃ of insulations 30 minutes, in water-bath, be heated to 95 ℃ then, keep annealing slowly after 3 minutes, naturally cool to room temperature.Two distrand DNA fragments after will annealing are again mixed (that is: (1+3)+(2+4)), and add following reagent:

10 * ligase enzyme damping fluid	2.5μl
10 * ligase enzyme damping fluid	2.5μl	ATP(0.1mol/L)	1μl
The T4 ligase enzyme	2μl	ATP(0.1mol/L)	1μl

Behind the mixing, keep to spend the night making it to connect in 16 ℃.Pass through 12%PAGE or 1% agarose gel electrophoresis then, ethidium bromide staining, with λ DNA/EcoRI, Sal I double digestion dna fragmentation or φ * 174DNA/SalI endonuclease bamhi is a molecular weight marker, divide the gene fragment of getting connection through extracting, recovery, drying, dissolve standby.

As shown in Figure 1, with the gene segment that obtains EcoR I/Sal I double digestion, thereby obtain sticky end.

The clone of GLP-1 gene

Get the plasmid 1 μ g (1 μ g/ml) that contains the Lac promotor, add 1 * 10 medium salt buffer, 1 μ l, freshly prepd distilled water 6 μ l, add each 1 μ l of restriction enzyme EcoRI, SalI then, in 37 ℃ of insulations 30 minutes, through chloroform-phenol reagent extracting, and with behind the chloroform washing water layer, use 60% isopropanol precipitating, centrifugal, dry back is standby.

Dissolve above-mentioned plasmid fragment with 4 μ l distilled waters, and mix, add 1 * 10 ligase enzyme damping fluid, 1 μ l, ATP 1 μ l, reach T4 ligase enzyme 2 μ l, spend the night in 16 ℃ of connections with the GLP-1 analogue gene fragment 2 μ l that are connected gained.

As shown in Figure 3, can be behind restriction restriction endonuclease BamH I/Sal I double digestion with the plasmid that obtains, be connected with said gene segment again through Bg III/Sal I double digestion, just can obtain containing dual-gene pulsating plasmid.Carry out this step repeatedly and can obtain containing 2-32 plasmid that copies.

Transform

Get single bacterium colony of an intestinal bacteria JM103, put in the LB nutrient solution and spend the night, therefrom get 500 μ l and be inoculated in the 100mlLB nutrient solution, in 37 ℃ of shaking culture, when cell concentration reaches A _600nmStop during=0.6 (0.D.).Centrifugal in 6000rpm, collect thalline, thalline is suspended in 2ml0.1mol/LMgCl ₂In the solution, in ice bath, kept 20 minutes.Centrifugal in 6000rpm again, collect thalline, be suspended in 2ml 0.1mol/LCaCl again ₂In the solution, put in the ice bath preserve standby; Or add 15% glycerine, freezing standby in-84 ℃.

Get above competent cell 100 μ l, add the Lac promoter plasmid that contains the GLP-1 analogue gene, in ice bath, kept 30 minutes, be transferred to 42 ℃ then and kept 2 minutes, cooling, coated plate (1% agar powder, contain 50 μ g/ml penbritins (AP)), 37 ℃ of overnight incubation; Picking colony is transferred to respectively in the test tube of the LB nutrient solution that contains 50 μ g/ml AP, and 37 ℃ of shaking culture are spent the night, and identifies in order to the extracting plasmid and uses.Meanwhile, each the single bacterium colony that is extracted is all lined on the LB-agar plate,, obtain engineering bacteria P lac/E8, the usefulness of reserving seed for planting (standby kind) through 37 ℃ of overnight incubation.

Fermentation

The LB nutrient solution is used in fermentation, contains peptone 10g, yeast powder 5g and NaCl 10g/L.

Inoculation P lac/E8 engineering bacteria ferments in 10 liters of fermented liquids, and condition is: stirring velocity 500rpm, air flow are tank volume of per minute, and 37 ℃, pH 7～8, dissolved oxygen 30～50%.The centrifugal collection thalline of 6000rpm after 20 hours that ferments, thalline is suspended in the 20mmol/L phosphoric acid buffer and (contains NaCl 1mol/L).Added N,O-Diacetylmuramidase by 1000: 1,30 ℃ are incubated 1 hour, carry out broken wall, centrifugal behind the broken wall (10,000rpm) collecting precipitation.Precipitate in the Guanidinium hydrochloride of the 6M that is suspended in 4 times of volumes, stir extracting and spend the night, 20,000rpm is centrifugal, and supernatant is dialysed to water, and 10,000 centrifugal collecting precipitations get inclusion body.Inclusion body weight in wet base 100g is suspended in the 1000ml water, and agitation condition drips 10ml 2-methylsuccinic acid acid anhydride down, constantly adds small amount of N a in the process ₂CO ₃Pulvis is kept solution PH 8-9, reacts 2 hours, makes it fully, and dialysis is desalted to water then.At PH7.0, add trypsin 1 in the 20mmole/L phosphoric acid buffer: 1000w/w) 37 ℃ of insulations cracking in 2 hours, regulate PH2-3 with 4mole/L hydrochloric acid, stir to keep and removed protecting group in 4 hours, through the HPLC purifying, gradient is the A phase: contain 0.5% aqueous formic acid; B phase: the solution that contains acetonitrile 80% (v/v) and formic acid 0.5%.A phase, B phase solution be with the linear gradient wash-out, 20ml/ minute flow velocity 280nm, the 220mM ultraviolet wavelength detects, pure product GLP-1 analogue.Whole process flow is seen Fig. 2.

Embodiment 2: produce G with gene engineering method ⁸-GLP-1 (7-35)-PPSR

1)5’-AAT TCC AGA TCT ATG CGT CAC GGT GAA GGT ACC TTCACC AGC GAT GTG AGC AGC TAT CTG-3’

2)5’-GAA GGT CAG GCG GCG AAA GAA TTT ATC GCG TGG CTGGTG AAA GGT CCG CCG AGC CGT GGA TCC TAG G-3’

3)5’-TGGAC CTA GGA TCC ACG GCT CGG CGG ACC TTT CACCAG CCA CGC GAT AAA TTC TTT ACC CGC CTG-3’

4)5’-ACC TTC CAG ATA GCT GCT CAC ATC GCT GGT GAA GGTACC TTC CCA GTG ACG CAT AGA TCT GG-3’

With 4 gene segments among above-mentioned 4 gene segments replacement embodiment 1, the separation of cloning process, fusion rotein, purifying, cracking are all identical with embodiment 1.

Embodiment 3, with the synthetic GLP-1 (7-35) of solid-phase synthesis-PPSR and G ⁸-GLP-1 (7-35)-PPSR

Chemical structure

(1)GLP-1(7-35)-PPSR

H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Pro-Pro-Ser-Arg-NH ₂

(2)G ⁸-GLP-1(7-35)-PPSR

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Pro-Pro-Ser-Arg-NH ₂

The selection of solid-phase resin

When peptide C-end is carboxyl

Select conventional king's resin (Wang Resin purchases in Shanghai gill biochemical corp) to carry out solid phase synthesis.Behind end of synthesis, the polypeptide resin of the band Side chain protective group that obtains is carried out cracking (be the deprotection base and cut off one step of resin and finish), and terminal just the fracture from king's resin of the C-of polypeptide forms carboxyl.

When peptide C-end is acid amides

Select conventional aminoresin (Fmoc-PAL-PEG-PS Resin purchases the company in PE AppliedBiosystem) to carry out solid phase synthesis.Behind end of synthesis, the polypeptide resin of the band Side chain protective group that obtains is carried out cracking (be the deprotection base and cut off one step of resin and finish), and terminal just the fracture from aminoresin of the C-of polypeptide forms acid amides.

Synthesis step

Fmoc-amino acid starting material → solid phase synthesis → take off Side chain protective group → HPLC purifying → lyophilize → GLP-1 (7-35)-PPSR/G of 17 kinds of band Side chain protective groups ⁸-GLP-1 (7-35)-PPSR

1. amino acid monomer

2. the process of solid phase synthesis

As shown in Figure 4, solid phase synthesis system is connected the terminal arginine of the C-of GLP-1 (7-35)-PPSR earlier on the aminoresin, and amino acid extends to the N-end from the C-end one by one then, carries out for 33 steps altogether.

(1) plant and instrument:

Applied Biosystem Peptide synthesizer, the 433A type

(2) reagent:

Wherein: DMF=N, dinethylformamide,

DIEA=N, the N-diisopropylethylamine,

The NMP=N-methyl-2-pyrrolidone,

The DMAP=Dimethylamino pyridine,

The HOBT=1-hydroxybenzotriazole,

HBTU=2-(1H-benzotriazole-Ji-1,1,3,3-tetramethyl--Uroniumhedrofluorophosphate.

(3) operation

With the 0.25mmole scale is example; take by weighing the about 0.5g of resin; insert in the reactor on the Peptide synthesizer; various band protecting group amino acid are taken by weighing the 1mmole bottling; aminoacid sequence by GLP-1 (7-35)-PPSR/G8-GLP-1 (7-35)-PPSR is arranged in the synthesizer to the N-end from the C-end, under 25 ℃ of room temperature conditions, is taken off Fmoc protection, activation automatically, is linked by computer program (SynthAssiat) control; by carrying out the next round circulation shown in the figure again, so carried out for 33 steps and finish synthetic.

In the amino acid condensation process, activated carboxylic generally need there be HBTU-Fast moc method, HOBt/DCC method, general DCC, methods such as acid anhydrides method.We adopt HBTU-Fast moc method to carry out activated carboxylic, as figure below.

Behind end of synthesis, the polypeptide resin of the band Side chain protective group that obtains is weighed after drying up on the Peptide synthesizer, then deprotection base and cut off one step of resin and finish.

3. deprotection base and cut off resin:

(1) plant and instrument

The magnetic agitation instrument

(2) reagent

GLP-1 (7-35)-PPSR/G with the band protecting group ⁸-GLP-1 (7-35)-PPSR polypeptide resin places tool plug Erlenmeyer flask, adds lytic reagent such as following table:

Under 30 ℃ of conditions of constant temperature, induction stirring was reacted 6 hours then; Filter, collect filtrate, resin washs with a small amount of trifluoroacetic acid; Merge and collect liquid and washings, (about 200～250mL) produce precipitation to the adding ether, filter, and precipitation is put into moisture eliminator after washing with a small amount of ether immediately.

4.HPLC separation and purification

Plant and instrument

Reagent

Preparing purity by two step HPLC reversed phase chromatographies is product more than 95%.

(1) the first step rough segmentation:

A). chromatographic column:

Adopt the reversed phase chromatography post of C18 medium.

B). moving phase:

A phase: contain 0.5% (v/v) acetic acid water solution;

B phase: contain second eyeball 80% (v/v) and 0.5% (v/v) acetic acid water solution;

C). go up sample solution:

The polypeptide crude product (purity about 30～50%) that polypeptide-resin is removed resin and Side chain protective group through cracking is made into the solution that concentration is 5.0mg/ml with 0.5% acetic acid solution.

D). Xian takes off condition:

Adopt linear gradient Xian to take off, the operation flow velocity is 20ml/min, and the ultraviolet detection wavelength is 280nm, and column temperature is a room temperature, and gradient is:

E). sample collection:

Xian who collects 20～60%B takes off main peak, product purity about 75～85%.

(2) second one-step refinings:

A). chromatographic column:

Adopt the reversed phase chromatography post of C18 medium.

B). moving phase: identical with the first step rough segmentation.

C). go up sample solution:

The distilled water that the collection liquid (purity about 75～85%) that the first step rough segmentation is obtained adds with volume dilutes.

D). elution requirement:

Employing segmentation Xian is one by one taken off, and the operation flow velocity is 20ml/min, and the ultraviolet detection wavelength is 280nm, and column temperature is a room temperature, and Xian takes off program and is:

B2 column volume → 35% a B2 column volume, 50% B6 column volume;

E). sample collection:

Xian who collects 50% B takes off liquid, the purity of product＞95%.

5. lyophilize

(1) plant and instrument

(2) operation

Earlier GLP-1 (7-35)-PPSR or G8-GLP-1 (7-35)-PPSR solution were put into cryogenic refrigerator freezing 2 hours, then it is put into and carry out freeze-drying in the freeze drying equipment and obtain required compound.

Embodiment 4, GLP-1 (7-35)-PPS-NH ₂Synthetic

Utilize Peptide synthesizer (Applied Biosystem) to be undertaken by embodiment 3 described solid phase synthesis process.

Embodiment 5, G ⁸-GLP-1 (7-35)-PPSOH's is synthetic

Carry out as method as described in the embodiment 4.

Embodiment 6, G ⁸-GLP-1 (7-35)-PPS-NH ₂Synthetic

Carry out as method as described in the embodiment 4.

Embodiment 7, G ⁸The hypoglycemic activity of-GLP-1 (7-35)-PPSR

Get C57 mouse (Chinese Academy of Sciences's Shanghai animal center), in 8 ages (body weight 20g) in week, 9, be divided into 3 groups.Only give drinking-water, overnight fasting.

Control group 0The time, an eye hole is got blood 30 μ l, places 0.3ml 0.9%NaCl solution, abdominal injection 20% glucose solution 200 μ l then, and respectively at 60,120, same blood sampling in 150,180 minutes, wherein abdominal injection 20% glucose 200 μ l for the second time in the time of 120 minutes.Blood sample after centrifugal 5 minutes, makes the blood cell precipitation through 3000rpm, gets whole centrifuged supernatant, and with health ministry, the blood sugar detection test kit that Shanghai Vaccine and Serum Institute produces carries out blood sugar detection.

GLP-1 (7-36)Group according to the same quadrat method of control group, got the blood pneumoretroperitoneum at 0 o'clock to inject 20% glucose solution, 200 μ l, subcutaneous injection GLP-1 (7-36) 2 μ g, and blood sugar detection carries out with quadrat method by control group.

G ⁸ -GLP-1 (7-35)-PPSRGroup is carried out with quadrat method by GLP-1 (7-36) group.

Three groups of hypoglycemic measurement results illustrate G shown in Fig. 5 A ⁸-GLP-1 (7-35)-PPSR has more long-lasting than GLP-1 (7-36).

The hypoglycemic activity of embodiment 8, GLP-1 (7-35)-PPSR and other compound

Press the hypoglycemic activity of embodiment 7, the results are shown in Figure 5B and 5C with quadrat method mensuration GLP-1 (7-35)-PPSR and other compound of the present invention.

Embodiment 9, G ⁸The promoting insulin secretion of-GLP-1 (7-35)-PPSR

C57 mouse (body weight 20g) overnight fasting, abdominal injection 40% glucose 100 μ l only give glucose for first group, subcutaneous injection physiological saline 100 μ l give sugar back subcutaneous injection GLP-1 analogue solution 100 μ l (2 μ g content of dispersion) for second group, in 0,2,5,10,20,30,60,90,120min gets blood 20 μ l, is blown in the centrifuge tube (to add 20 μ l EDTA 1% in advance), and centrifuging and taking serum is surveyed Regular Insulin (Fig. 6) with Regular Insulin test kit (Crystal Chem Inc).

Claims

1, GLP-1 analogue, its structural formula is as follows:

HX ₁EGTFTSDVSSYLEGQAAKEFLAWLVKGPPSX ₂

X ₁=Ala or Gly,

X ₂=Arg-OH, OH or NH ₂

2. GLP-1 analogue as claimed in claim 1, wherein said X ₁Be Gly.

3. GLP-1 analogue as claimed in claim 1, wherein said X ₁Be Ala.

4. as each described GLP-1 analogue of claim 1-3, wherein said X ₂Be Arg-OH.

5. as each described GLP-1 analogue of claim 1-3, wherein said X ₂Be OH.

6. as each described GLP-1 analogue of claim 1-3, wherein said X ₂Be NH ₂

7. pharmaceutical composition, it contains each described GLP-1 analogue just like claim 1-6.

8. pharmaceutical composition as claimed in claim 7, it also contains pharmaceutically acceptable thinner, vehicle or carrier.

9. pharmaceutical composition as claimed in claim 8, wherein said carrier are selected from as next group material: ethanol, glycerine, water.

10. as the purposes of each described GLP-1 analogue of claim 1-6 in the medicine of preparation treatment diabetes.

11. purposes as claimed in claim 10, wherein said diabetes are H type diabetes.

12. as the purposes of each described pharmaceutical composition of claim 7-9 at preparation treatment diabetes medicament.

13. purposes as claimed in claim 12, wherein said diabetes are type ii diabetes.