CN103193881A - Hypoglycemic polypeptide derivative for oral medication and use thereof - Google Patents
Hypoglycemic polypeptide derivative for oral medication and use thereof Download PDFInfo
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- CN103193881A CN103193881A CN201310137942XA CN201310137942A CN103193881A CN 103193881 A CN103193881 A CN 103193881A CN 201310137942X A CN201310137942X A CN 201310137942XA CN 201310137942 A CN201310137942 A CN 201310137942A CN 103193881 A CN103193881 A CN 103193881A
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Abstract
The invention relates to a hypoglycemic polypeptide derivative for oral medication and use thereof, and belongs to the field of medicinal biotechnology. A polypeptide part of a polypeptide derivative-1 with hypoglycemic activity has an amino acid sequence of SQD ID NO:1 in sequence table; the polypeptide part table of a polypeptide derivative-2 has an amino acid sequence of SQD ID NO:2 in the sequence table; and the N-terminal amino acid residue or a lysine residue side chain or a cysteine side chain of the polypeptide are connected with a substituent group. By adopting the hypoglycemic polypeptide derivative, two protein polypeptide derivatives are successfully constructed for the first time; the defects of large pains on body and mind of a sufferer due to the fact that the drug is short in half-life period and must be frequently injected for a long period of time are overcome; and the polypeptide derivative can be used for oral medication, and exerts the hypoglycemic activity. Therefore, on one hand, the security is improved; on the other hand, the pain to the patient caused by parenteral administration is reduced; the compliance on the patient is improved; and long-term stable control of blood sugar is also facilitated.
Description
Technical field
The present invention relates to the preparation and uses thereof of the Polypeptide-k derivative of Orally-administrable, specifically the active polypeptide with hypoglycemic activity is carried out structure of modification and modification, make its acquisition can absorb and reach in vivo the characteristic of effective treatment concentration by gi tract, and this polypeptide derivative is used for treatment of diabetes, belong to the medical biotechnology field.
Background technology
Diabetes are to be common disease, the frequently-occurring disease of feature with the chronic hyperglycemia, be by insulin secretion in the body or effect defective, or the two exist and the sugar, fat, the protein metabolism disorder that cause simultaneously.Mainly contain two types of insulin-dependent (IDDM, 1 type) and non-insulin-depending types (NIDDM, 2 types) clinically.Along with standard of living improves, the sickness rate of diabetes increases year by year, has become the chronic disease of the 3rd serious harm human health after tumour, cardiovascular and cerebrovascular diseases.Present global number of patients has reached 1.5 hundred million, wherein is the diabetes B patient more than 90%
[1]According to World Health Organization prediction, to the year two thousand thirty the whole world 300,000,000 diabetes B patients will be arranged.Diabetes B patient's traditional treatment pattern generally is to follow the staged treatment of dietary control, oral antidiabetic thing and exogenous insulin.But the treating diabetes field still exists many major issues that waits to solve at present, seldom can control glucose level ideally as most methods for the treatment of, but also have some side effects and restriction.
(Glucagon-like peptide-1 GLP-1) is incretin by enteron aisle endocrine cell-L emiocytosis to glucagon-like-peptide-1, is Proglucagon gene translation post-treatment product, and multiple existence form is arranged in vivo.The GLP-1 of intestinal secretion (1～37) is the peptide chain of non-activity, and N holds 6 peptides through the enzymolysis excision, becomes the GLP-1 (7～37) of biologically active.Its C-terminal glycine can be used as the substrate of amidating enzyme, and the C-terminal amidation has increased the body internal stability of GLP-1.The aminoacid sequence of GLP-1-(7～36)-NH2 and GLP-1-(7～37) is as follows:
GLP-1-(7～36)-NH
2：H
7AEG
10TFTSD
15VSSYL
20EGQAA
25KEFIA
30WLVKG
35R-NH
2
GLP-1-(7～37)：H
7AEG
10TFTSD
15VSSYL
20EGQAA
25KEFIA
30WLVKG
35RG-COOH
The major physiological effect of GLP-1 comprises: the release action of (1) glucose dependency pancreotropic hormone.Its mechanism of action be by with the special acceptor interaction on pancreatic beta cell surface, the insulin secretion of glucose induction is significantly increased.(2) the stimulating growth chalone discharges the secretion of sharp glucagon suppression.(3) suppress parietal cell secretion hydrochloric acid in gastric juice, the emptying that prolongs stomach.(4) increase satiety, depress appetite reduces the picked-up of energy.(5) strengthen beta Cell of islet function and promote its propagation.GLP-1 is as the remarkable advantage of diabetes B medicine: the effect of the sharp glucagon suppression secretion of the promoting insulin secretion of (1) GLP-1 all depends on the concentration of glucose, hypoglycemia can not take place with its treatment diabetes; (2) GLP-1 can increase patient's satiety owing to suppress the stomach emptying, suppresses patient's appetite, thereby loses weight.
Although natural GLP-1 has plurality of advantages in the treatment diabetes, the transformation period only is about 2 minutes in its body, has limited its direct application clinically.But this is owing in the body a kind of DPP IV (DPP-IV) being arranged the active deputy Pro of peptide N-terminal of adjusting of specific recognition or Ala residue, from the N-terminal excision dipeptides of peptide chain.And the N-terminal second amino acids residue of GLP-1 is Ala just, makes GLP-1 be degraded to GLP-1 (9～36)-NH2 and the GLP-1 (9～37) of non-activity in vivo very soon.Studies show that, to the terminal primary His of GLP-1N
7Or deputy Ala
8Modify the degraded that can prevent DPP-IV, but work as His
7But might cause GLP-1 to lose biologic activity after being modified, delete or otherwise being substituted; Deputy L-Ala
8Being replaced by D-Ala or being replaced the back by other small molecules amino acid such as Gly is not had influence to its biologic activity, and this just provides foundation for GLP-1 derivative or analogue research.
At present, there is Many researchers to construct GLP-1 analogues such as the chemically modified analogue of GLP-1 or fusion rotein, some of them GLP-1 analogue can be simulated the effect of endogenous GLP-1, is the novel ofhypoglycemic medicine that a class has multiple anti-high-blood-sugar function, as the Liraglutide that has gone on the market
[13]And Exenatide
[14], also have some analogues to be in clinical experimental stage, for example CJC1131 (conjuchem), Albugon and ZP10A (zealand pharma and aventis) etc.
Because the character of polypeptide drug itself and human body are to the various barriers of its generation, its conventional route of administration is always based on injection.Because this class drug half-life is short, frequent injection for a long time causes the huge misery of patient on body ﹠ mind.Therefore, development polypeptide drug non-injection administration approach is current the world of medicine Focal Point of Common Attention, and wherein, oral administration becomes developing direction because of convenient, safety.
Summary of the invention
Have the endogenous cell movement system in the gi tract, coupling can by receptor mediated endocytosis, can be increased the intestinal absorption of protein and polypeptide by the transhipment-carrier molecule of this system identification on protein and polypeptide.Receptor mediated endocytosis can not be subjected to limiting by the drug molecule size of cell, can be comprised Sugar receptors class, endotoxin, Transferrins,iron complexes and vitamins (vitamins B by the part that acceptor is identified
12, folic acid, riboflavin and vitamin H) etc.
Vitamin H (biotin) is the maximum a kind of water-soluble vitamins of Recent study, has the important physical function in animal body.Bacterium, yeast, mould, algae and some plants can synthesizing biotinylated, and people and other Mammalss can not synthesizing biotinylated, can only absorb from food.Exist vitamin H active absorption mechanism in the animal body, participate in the carrier of vitamin H active absorption and transhipment for relying on Na
+Poly-vitamin transport vehicle (SMVT, sodium dependent multivitamin transporter).The researchist finds that hamster, people, rat, rabbit vitamin H exists active absorption, i.e. SMVT in small intestine.SMVT is the main movement system of vitamin H picked-up, and SMVT can also transport pantothenic acid and Thioctic Acid except the transhipment vitamin H
[19]Enteron aisle by the characteristics of SMVT active transport organism-absorbing element is: 1. the transhipment meeting of vitamin H is suppressed (desthiobiotin, biotin methyl ether, diamines vitamin H and biotin complex of yeast) by its analog; 2. rely on Na
+3. power consumption; 4. rely on certain temperature; 5. carry out in serous coat contrary concentration gradient in (serosal compartment)
[20]
The medicine that transports low intestinal permeability based on SMVT has caused people's attention, with medicine and vitamin H coupling, by SMVT intestinal epithelial cell is gone in drug transport, can improve the intestinal mucosa transmitance of medicine.Therefore, the vitamin H active absorption that can utilize SMVT to mediate is improved the oral administration biaavailability of polypeptide drug.
The present invention proposes a kind of SMVT that can utilize and mediates the oral polypeptide derivative with hypoglycemic activity, its polypeptide portion is to pass through the polypeptide with hypoglycemic activity that changes structure and obtain at GLP-1 and Exendin-4 basis, and the side chain of polypeptide amino acid residue is connected with the substituting group that can be combined with SMVT.This derivative is combined with SMVT by substituting group, and the transhipment of guiding polypeptide portion enters intestinal epithelial cell, reaches effective treatment Plasma Concentration, the performance hypoglycemic activity.
The invention has the advantages that: this polypeptide derivative can pass through oral administration, and the performance hypoglycemic activity has improved security on the one hand, on the other hand, reduce drug administration by injection to the misery that patient brings, improved patient's compliance, be conducive to control blood sugar steady in a long-term.
Description of drawings
The SDS-PAGE detected through gel electrophoresis result of Fig. 1 Polypeptide-k derivative-1.The M swimming lane is represented Marker, and 1 swimming lane represents that Polypeptide-k-1,2 swimming lane represents Polypeptide-k derivative-1.
The SDS-PAGE detected through gel electrophoresis result of Fig. 2 Polypeptide-k derivative-2.The M swimming lane is represented Marker, and 1 swimming lane represents that Polypeptide-k-2,2 swimming lane represents Polypeptide-k derivative-2.
The oral hypoglycemic activity of Fig. 3 Polypeptide-k derivative-1 and derivative-2 is measured.
Embodiment
Below in conjunction with embodiment, be described in further detail the present invention.The bacterial strain that adopts among specification sheets and the embodiment, plasmid, chemical reagent etc., as specified otherwise not all routinely experiment condition operate, or operate by the explanation that supplier provides.
The preparation of embodiment 1 Polypeptide-k-1:
The first step: according to the aminoacid sequence of Polypeptide-k mutant (SQD ID NO:1), with reference to the synthesizing single-stranded DNA of e. coli codon preferences, and at upstream interpolation EK restriction enzyme site (DDDDK), the recognition sequence of KpnI and HindIII is added at two ends respectively.
Second step: synthetic gene is connected with the pET32a carrier, carry out double digestion with KpnI and HindIII respectively, product after enzyme is cut connects 8 hours for 16 ℃, and the standard method that provides according to " molecular cloning " will connect product transformed into escherichia coli competent cell and select positive colony.Sequence verification is inserted the accuracy of gene.
The 3rd step: 37 ℃ of positive colonies are spent the night with the LB culture medium culturing do seed liquor, add 50 μ g/mL kantlex in the substratum and do selection pressure.By 1% inoculum size seed liquor is forwarded to 100mL LB substratum, 37 ℃ are cultured to OD600 and reach 0.6-1.0, add 0.2-1mmol/LIPTG and induce, and finish after inducing culture 4-10 hour to cultivate.
The 4th step: 4000 change 20 minutes centrifugal results bacterial sediments, carry out chromatography purification with Ni-IMAC behind the supersonic wave wall breaking, carry out chromatography purification with Ni-IMAC again after cutting with enteropeptidase, obtain Polypeptide-k-1, and electrophoresis detection the results are shown in accompanying drawing 1.
The preparation of embodiment 2 Polypeptide-k derivatives-1:
The first step: the vitamin H succinimide is dissolved in 1mg is dissolved in (pH=1) in the 1ml dilute acetic acid, get Polypeptide-k-15mg and be dissolved in 1ml PB damping fluid (pH=8.8).
Second step: will appeal 2: 1 in molar ratio mixed of solution, reaction is spent the night.
The 3rd step: utilize Q anionite-exchange resin purifying, obtain Polypeptide-k derivative-1.Electrophoresis detection the results are shown in accompanying drawing 1.
The preparation of embodiment 3 Polypeptide-ks-2:
The first step: according to the aminoacid sequence of Polypeptide-k mutant (SQD ID NO:2), with reference to the synthesizing single-stranded DNA of e. coli codon preferences, and at upstream interpolation EK restriction enzyme site (DDDDK), the recognition sequence of KpnI and HindIII is added at two ends respectively.
Second step: synthetic gene is connected with the pET32a carrier, carry out double digestion with KpnI and HindIII respectively, product after enzyme is cut connects 8 hours for 16 ℃, and the standard method that provides according to " molecular cloning " will connect product transformed into escherichia coli competent cell and select positive colony.Sequence verification is inserted the accuracy of gene.
The 3rd step: 37 ℃ of positive colonies are spent the night with the LB culture medium culturing do seed liquor, add 50 μ g/mL kantlex in the substratum and do selection pressure.By 1% inoculum size seed liquor is forwarded to 100mL LB substratum, 37 ℃ are cultured to OD600 and reach 0.6-1.0, add 0.2-1mmol/LIPTG and induce, and finish after inducing culture 4-10 hour to cultivate.
The 4th step: 4000 change 20 minutes centrifugal results bacterial sediments, carry out chromatography purification with Ni-IMAC behind the supersonic wave wall breaking, carry out chromatography purification with Ni-IMAC again after cutting with enteropeptidase, obtain Polypeptide-k-2.Electrophoresis detection the results are shown in accompanying drawing 2.
The preparation of embodiment 4 Polypeptide-k derivatives-2:
The first step: the vitamin H succinimide is dissolved in 1mg is dissolved in (pH=1) in the 1ml dilute acetic acid, get Polypeptide-k-25mg and be dissolved in 1ml PB damping fluid (pH=8.8).
Second step: will appeal 2: 1 in molar ratio mixed of solution, reaction is spent the night.
The 3rd step: utilize Q anionite-exchange resin purifying, obtain Polypeptide-k derivative-2.Electrophoresis detection the results are shown in accompanying drawing 2.
The oral hypoglycemic activity of embodiment 5 Polypeptide-k derivatives-1 and Polypeptide-k derivative-2 is measured:
Choose 24 of male KM mouse, measure fasting plasma glucose, the mouse that fasting blood sugar is approached is divided into one group, totally 4 groups, is respectively high sugared group, control group and administration group.High sugar group is pressed 18mmol/kg dosage abdominal injection glucose, and the administration group is then pressed 50nmol/kg dissolving Polypeptide-k derivative-1 and Polypeptide-k derivative-2 back administration in the glucose solution with dosage, and control group only gives physiological saline.The administration volume is all controlled at 8mL/kg.Measure blood-sugar content respectively at 0 minute, 15 minutes, 30 minutes, 60 minutes and 90 minutes blood sample collections after the administration.The results are shown in accompanying drawing 3.
Claims (3)
1. polypeptide derivative with hypoglycemic activity, its polypeptide portion has the aminoacid sequence shown in sequence table SQD ID NO:1 or the sequence table SQD ID NO:2, the N-terminal amino-acid residue of polypeptide or lysine residue side chain, or cysteine side chain is connected with substituting group.
2. the described polypeptide derivative of claim 1 is characterized by described substituting group and has following structure:
Wherein, n=1～6, substituting group is connected with the α amino of polypeptide N-terminal amino-acid residue or the amino amido linkage that forms of ε of lysine residue side chain.The polypeptide derivative that forms has following structure:
。
3. the described polypeptide derivative of claim 1 is for the preparation of the orally-taken blood sugar reducing medicine.
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| CN201310137942XA CN103193881A (en) | 2013-04-22 | 2013-04-22 | Hypoglycemic polypeptide derivative for oral medication and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103641889A (en) * | 2013-12-12 | 2014-03-19 | 中国药科大学 | Hypoglycemic peptide and drug use thereof |
| CN109485720A (en) * | 2017-09-11 | 2019-03-19 | 中国药科大学 | Oral hypoglycaemic polypeptide, its fatty acid modifying derivative and purposes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724563A (en) * | 2005-06-29 | 2006-01-25 | 常州制药厂有限公司 | Exendin 4 polypeptide segment |
| CN101328221A (en) * | 2008-04-14 | 2008-12-24 | 中国药科大学 | Hypoglycemic polypeptide fused protein, structure and use of derivate thereof |
| CN102712690A (en) * | 2009-11-26 | 2012-10-03 | 吴晓琰 | Long-acting Exendin 4 analogues |
-
2013
- 2013-04-22 CN CN201310137942XA patent/CN103193881A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724563A (en) * | 2005-06-29 | 2006-01-25 | 常州制药厂有限公司 | Exendin 4 polypeptide segment |
| CN101328221A (en) * | 2008-04-14 | 2008-12-24 | 中国药科大学 | Hypoglycemic polypeptide fused protein, structure and use of derivate thereof |
| CN102712690A (en) * | 2009-11-26 | 2012-10-03 | 吴晓琰 | Long-acting Exendin 4 analogues |
Non-Patent Citations (2)
| Title |
|---|
| CHENG-HAO JIN等: "A new orally available glucagon-like peptide-1 receptor agonist, biotinylated exendin-4, displays improved hypoglycemic effects in db/db mice", 《JOURNAL OF CONTROLLED RELEASE》 * |
| 毕华等: "Exendin-4的研究进展", 《药物分析杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103641889A (en) * | 2013-12-12 | 2014-03-19 | 中国药科大学 | Hypoglycemic peptide and drug use thereof |
| CN103641889B (en) * | 2013-12-12 | 2016-02-10 | 中国药科大学 | A kind of incretin peptide and pharmaceutical use thereof |
| CN109485720A (en) * | 2017-09-11 | 2019-03-19 | 中国药科大学 | Oral hypoglycaemic polypeptide, its fatty acid modifying derivative and purposes |
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Application publication date: 20130710 |