CN1746185A - Analog of Exendin 4 - Google Patents

Analog of Exendin 4 Download PDF

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Publication number
CN1746185A
CN1746185A CN 200410054300 CN200410054300A CN1746185A CN 1746185 A CN1746185 A CN 1746185A CN 200410054300 CN200410054300 CN 200410054300 CN 200410054300 A CN200410054300 A CN 200410054300A CN 1746185 A CN1746185 A CN 1746185A
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diabetes
exendin
analogues
exendin4
analogue
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CN100535003C (en
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孙玉琨
张培璋
周波
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Shanghai Benemae Pharmaceutical Corp
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Huayi Bio-Technology Co Ltd Shanghai
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Abstract

Polypeptide as Exendin4 analogs and its production by chemical synthesis and recombinant DNA are disclosed. The analogs can be used to decrease blood sugar and treat II type diabetes.

Description

The analogue of Exendin4
Invention field
The present invention relates to the polypeptide of Exendin4 analogue, this polypeptide has the lowering blood glucose effect, 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.
Exendin4 and Exendin3 are that huge lizard (Gila monster is produced in South America, HelodermeSuspectum) excretory two peptide species in the saliva, wherein Exendin 4 is made up of 39 amino-acid residues, and its structure and GLP-1 have 50% homology on aminoacid sequence.
Studies show that Ex4 combines with the acceptor of GLP-1 as the analogue of GLP-1.Animal and type ii diabetes patient's clinical 1,2,3 phases evidence, Ex4 can promote insulinogenic synthetic, insulin secretion accelerating, lowering blood glucose, no longer continuation effect after blood sugar is normal, thereby do not produce hypoglycemic coma, shock, fool proof.It can reduce HbAlc, increases the beta cell amount, increases the susceptibility of type ii diabetes patient insulin receptor, effects such as glucagon suppression secretion.
Compare with GLP-1, the clinical application of Ex4, dosage is every day 2 times, each 5-10 μ g, and GLP-1 then is about 200 μ g, and Ex4 is not subjected to the effect of DPPIV pepx in blood, transformation period is longer than GLP-1, but also has weak point, and Ex4 derives from huge lizard, and GLP-1 is people's a endogenous substance, and clinical experiment report Ex4 has 52% patient to produce antibody (Diabetes (1997) 46, 433-439; Diabetes Care (2002) 25, 330-336; JAMA (2002) 287, 373-379; Diabetes (1995) 44, 1249-1258; Diabetes (2002) 512796-2803; Diabetes Care (2000) 2364-69, N.Engl.J.Med (2002) 346, 393-463, Lancet (1998) 352, 837-853).
The research that changes structure about Ex4 begins to shorten peptide chain from the C-end, and the result shows that 9 amino-acid residues of C-end of Ex4 are active necessary, constantly shortens the peptide chain result from the beginning of N-end and shows and remove an amino acid His that activity influence is little.Yet these results are that isolated experiment obtains, and with the size of GLP-1 receptor binding capacity, the formation amount of what and cAMP of short insulinoma cell excreting insulin is estimated and had limitation (J.Biol.Chem. (1997) 272, 21201-21206; Regulatory Peptides (2003) 114, 153-158; Trend inpharmacological Sci. (2003) 24, 377-383; PCT, WO03/011892, A2).
The object of the invention is to change the chemical structure of Exendin4, shorten its peptide chain length and change amino acid structure from the C end, keep its hypoglycemic activity, thereby the homology of the aminoacid sequence of raising and Exendin 4, structurally satisfy simultaneously the requirement that chemosynthesis and gene engineering method are produced, suitable a large amount of the manufacturing reduces cost, and adapts to vast type ii diabetes patient's needs.The present invention does not adopt stripped cAMP to generate, secretion of insulin and GLP-1 receptor binding capacity method, and directly adopt the lowering blood glucose method that the Exendin4 analogue is estimated, help instructing Clinical Application.
Summary of the invention
The invention provides following Exendin4 analogue:
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKN 28PPSX 3 Em32
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKNG 29PPSX 3 Em33
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKNGG 30PPSX 3 Em34
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKNGGP 31PPSX 3 Em35
HGEGTFTSDLSKQX1EEEAVX2LFIEWLKNGGPSPPSX3 Em36
HGEGTFTSDLSKQX1EEEAVX2LFIEWLKNGGPSSPPSX3 Em37
HGEGTFTSDLSKQX1EEEAVX2LFIEWLKNGGPSSGPPSX3 Em38
X 1=Met, Leu or Ile;
X 2=Lys;
X 3=Arg, OH or NH 2
The present invention also provides the drug regimen that contains above-mentioned analogue and has prepared the method for above-mentioned Exendin4 analogue in addition, and making to vast type ii diabetes patient provides the Exendin4 medicine to become possibility in a large number.
Description of drawings
Fig. 1 is a synthetic Exendin4 analogue gene segment sequence chart;
Fig. 2 is the schema that gene engineering research is produced the Exendin4 analogue;
Fig. 3 is the structure synoptic diagram that contains the plasmid of multiple copied Exendin4 gene;
Fig. 4 is the synoptic diagram with the synthetic Em32 of solid-phase synthesis;
Fig. 5 is the HPLC analytical structure of Exendin4 analogue of the present invention;
The blood sugar decreasing effect figure of Fig. 6 A-B Exendin4 analogue of the present invention;
Fig. 7 is the insulin secretion accelerating design sketch of Exendin4 analogue of the present invention.
Detailed Description Of The Invention
One aspect of the present invention provides structure to change, and still has the Exendin4 analogue of hypoglycemic activity, and it has aforesaid structural formula.Preferably, X 1Be Met or Leu, X 2Be Lys.More preferably, X 1Be Met, X 3Be Arg.
Another aspect of the present invention provides the pharmaceutical composition that contains above-mentioned Exendin4 analogue, said composition contains one or more compounds with said structure formula and a kind of pharmaceutically acceptable thinner or vehicle, preferably said composition is a unit dosage form, as tablet, pill, capsule (comprise and continue to discharge or postpone releasing pattern), pulvis, particle, elixir, tincture, syrup and emulsion, disinfectant injection solution or suspension, 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 support of nontoxic pharmacy, as ethanol, glycerine, water and analogue 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 Exendin4 analogue, comprises chemical synthesis process and gene engineering method.For X 3Be the above-claimed cpd of Arg, can synthesize, can also synthesize by chemical process, but preferably synthesize with gene engineering method with gene engineering method.For X 3Be OH or NH 2Above-claimed cpd, can synthesize by chemical process.Preferred chemical process is a solid-phase synthesis.
Embodiment 1: produce HGEGTFTSDLSKQMEEEAVKLFI EWLKN with gene engineering method PPSR 32
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 EcoRI, 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 ATG AGA TCT CGT CAC GGT GAA GGT ACC TTCACC AGC GAT CTG AGC AAA CAG CTG GAA G
(2)5’-AA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG AAAAAC CCG CCG AGC CGT GGA TCC TAG G’
(3)5’-TTC TTC TTC CAG CTG TTT GCT CAG ATC GCT GGT GAAGGT ACC TTC ACC GTG ACG AGA TCT CAT GG
(4)5’-TC GAC CTA GGA TCC ACG GCT CGG CGG GTT TTT CAGCCA TTC GAT GAA CAG TTT AAC CGC
(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
Distilled water 3μl
The T4 polynucleotide kinase 1μl
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
ATP(0.1mol/L) 1μl
The T4 ligase enzyme 2μl
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/EcoR I, Sal I double digestion dna fragmentation or φ * 174DNA/Sal I 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 Exendin4 analogue 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, Sal I 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 Exendin4 analogue gene fragment 2 μ l that are connected gained.
As shown in Figure 3, the plasmid that obtains behind restriction restriction endonuclease BamH I/Sal I double digestion, is connected with said gene segment through Bg III/Sal I double digestion again, just can obtains containing dual-gene pulsating plasmid.Carry out the plasmid that this step can obtain containing 8 tandem copies repeatedly.
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 Exendin4 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 PE8/JM103 (containing 8 copies), 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 PE8/JM103 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 ℃, pH7~8, 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.
X of the present invention 3For other compound of Arg is all used as method preparation identical as described in the embodiment 1.
Embodiment 2, with the synthetic Em32 of solid-phase synthesis
Chemical structure
Em32 HGEGTFTSDLSKQ LEEEAVKLFIEWLKN PPS-OH
HGEGTFTSDLSKQ LEEEAVKLFIEWLKN PPS-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.
Preparation process
Fmoc-amino acid starting material → solid phase synthesis → take off Side chain protective group → HPLC purifying → lyophilize → Em32 of 17 kinds of band Side chain protective groups
1. amino acid monomer
The amino acid title The source
Fmoc-L-Ala-OH Fmoc-L-Arg(Pmc)-OH Fmoc-L-Asp(OtBu)-OH Fmoc-L-Gln(Trt)-OH Fmoc-L-Glu(OtBu)-OH Fmoc-L-Gly-OH Fmoc-L-His(Trt)-OH Fmoc-L-Ile-OH Fmoc-L-Leu-OH Fmoc-L-Lys(Trt)-OH Fmoc-L-Phe-OH Fmoc-L-Pro-OH Fmoc-L-Ser(tBu)-OH Fmoc-L-Thr(tBu)-OH Fmoc-L-Trp-OH Fmoc-L-Tyr(tBu)-OH Fmoc-L-Val-OH Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. PE Applied.Biosystem Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc. Peptide Institute,Inc.
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 Em32 earlier on the aminoresin (Fmoc-PAL-PEG-PS resin), 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:
Title Specification
N-Methyl pyrrolidone methylene dichloride hexahydropyridine 2.0M DIEA/NMP DMAP/DMF is dissolved in the HBTU 100mmole/0.5M HOBT of DMF The synthetic level of the synthetic level of polypeptide analytical pure analytical pure polypeptide synthetic level polypeptide synthetic level polypeptide
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 Em32s 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,
Figure A20041005430000151
The 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: magnetic agitation instrument
(2) reagent
Title Specification
Water trifluoroacetic acid phenol Deionized water analytical pure analytical pure
Mercaptoethanol methyl-phenoxide ether Analytical pure analytical pure analytical pure
The Em32s polypeptide resin of band protecting group is placed tool plug Erlenmeyer flask, adds lytic reagent such as following table:
Reagent Consumption (ml)
Water methyl-phenoxide phenol mercaptoethanol trifluoroacetic acid 0.50 0.50 0.75 0.20 10.0
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, adding ether (about 200-250mL) produces precipitation, filters, and precipitation is put into moisture eliminator after washing with a small amount of ether immediately.
4.HPLC separation and purification
Plant and instrument
Title Model
Rotary Evaporators analysis mode HPLC preparation HPLC ultraviolet flow detection record ZFQ85A LC10AD LC8A SIBAS,YOKOGAWA 3057
Reagent
Title Specification
Acetonitrile The HPLC level
Ethanol methyl alcohol trifluoroacetic acid HPLC level HPLC level HPLC level
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 (the about 30-50% of purity) 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:
Time (min) Mobile phase B
0 30 50 20% 60% 90%
E). sample collection:
Xian who collects 20-60%B takes off main peak, the about 75-85% of product purity.
(2). second one-step refining:
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 (the about 75-85% of purity) that the first step rough segmentation is obtained adds with volume dilutes.
D). Xian takes off condition:
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: 30%B2 column volume 35%B2 column volume 50%B6 column volume;
E). sample collection:
Xian who collects 50%B takes off liquid, the purity of product>95%.The result as shown in Figure 5.
5. lyophilize
(1) plant and instrument
Equipment Model
The vacuum freeze drier cryogenic refrigerator FD-5
(2) operation
Earlier Em32 solution was 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.
X of the present invention 3Be OH or NH 2Other compound all use as method preparation identical as described in the embodiment 2.
The lowering blood glucose effect of embodiment 3:Exendin4 analogue
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.
During control group 0, the eye hole is got blood 30 μ l, places 0.3ml 0.9%NaCl solution, abdominal injection 20% glucose solution 200 μ l then are 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.
Ex4 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 Exendin4 analogue 2 μ g, and blood sugar detection carries out with quadrat method by control group.
The Em32-38 group is carried out with quadrat method by the Ex4 group.
Three groups of hypoglycemic measurement results are shown in Fig. 6 A and B.
The promoting insulin secretion of embodiment 4:Exendin4 analogue
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 Exendin4 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 with Regular Insulin test kit (Crystal Chem Inc).The results are shown in Figure 7.

Claims (14)

1, Exendin 4 analogues, it has following structural formula:
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKN 28PPSX 3 Em32
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKNG 29PPSX 3 Em33
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKNGG 30PPSX 3 Em34
HGEGTFTSDLSKQX 1EEEAVX 2LFIEWLKNGGP 31PPSX 3 Em35
Wherein: X 1=Met, Leu or Ile;
X 2=Lys;
X 3=Arg, OH or NH 2
2, Exendin 4 analogues as claimed in claim 1, wherein said X 1Be Met.
3, Exendin 4 analogues as claimed in claim 1, wherein said X 1Be Leu.
4, Exendin 4 analogues as claimed in claim 1, wherein said X 1Be Ile.
5, as each described Exendin 4 analogues of claim 1-4, wherein said X 3Be R.
6, as each described Exendin 4 analogues of claim 1-4, wherein said X 3Be OH.
7, as each described Exendin 4 analogues of claim 1-4, wherein said X 3Be NH 2
8, a kind of pharmaceutical composition, it contains each described Exendin 4 analogues just like claim 1-7.
9, pharmaceutical composition as claimed in claim 8, it also contains pharmaceutically acceptable thinner, vehicle or carrier.
10, pharmaceutical composition as claimed in claim 8, wherein said carrier is selected from: as ethanol, glycerine, water.
11, as the purposes of each described Exendin 4 analogues of claim 1-7 in preparation treatment diabetes medicament.
12, purposes as claimed in claim 11, diabetes wherein are type ii diabetes.
13, as the purposes of each described drug regimen of claim 8-10 in preparation treatment diabetes medicament.
14, purposes as claimed in claim 13, diabetes wherein are type ii diabetes.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1908778A1 (en) * 2005-06-29 2008-04-09 Changzhou Pharmaceutical Factory Co., Ltd. Exendin 4 polypeptide fragments and use thereof
WO2011063549A1 (en) * 2009-11-26 2011-06-03 Wu Xiaoyan Long-acting exendin 4 analogues
CN101280012B (en) * 2007-03-14 2011-10-19 长春百克生物科技股份公司 Exendin-4 active isomer and application thereof
CN103613657A (en) * 2013-11-28 2014-03-05 孙玉琨 Exendin4 with shortened peptide chain and genetic engineering application thereof

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1908778A1 (en) * 2005-06-29 2008-04-09 Changzhou Pharmaceutical Factory Co., Ltd. Exendin 4 polypeptide fragments and use thereof
EP1908778A4 (en) * 2005-06-29 2009-08-12 Changzhou Pharmaceutical Facto Exendin 4 polypeptide fragments and use thereof
CN101280012B (en) * 2007-03-14 2011-10-19 长春百克生物科技股份公司 Exendin-4 active isomer and application thereof
WO2011063549A1 (en) * 2009-11-26 2011-06-03 Wu Xiaoyan Long-acting exendin 4 analogues
CN102712690A (en) * 2009-11-26 2012-10-03 吴晓琰 Long-acting Exendin 4 analogues
CN102712690B (en) * 2009-11-26 2016-04-13 吴晓琰 The analogue of long-acting Exendin 4
CN103613657A (en) * 2013-11-28 2014-03-05 孙玉琨 Exendin4 with shortened peptide chain and genetic engineering application thereof
CN103613657B (en) * 2013-11-28 2016-01-13 孙玉琨 Shorten Exendin4 and the genetically engineered application thereof of peptide chain

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