CN103012555A - Preparation method for new polypeptide having tissue protection activity, and application of new polypeptide in treatment - Google Patents
Preparation method for new polypeptide having tissue protection activity, and application of new polypeptide in treatment Download PDFInfo
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- CN103012555A CN103012555A CN2011102804884A CN201110280488A CN103012555A CN 103012555 A CN103012555 A CN 103012555A CN 2011102804884 A CN2011102804884 A CN 2011102804884A CN 201110280488 A CN201110280488 A CN 201110280488A CN 103012555 A CN103012555 A CN 103012555A
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Abstract
The present invention discloses a new polypeptide having a tissue protective activity. The present invention discloses a sequence, a structure and a preparation method of the polypeptide. The polypeptide has a good tissue and organ protection effect, especially provides good cure effects for inflammations of a plurality of tissues, and has characteristics of no toxicity, safety, and wide medicinal and medical application prospects.
Description
Technical field
The invention belongs to the medical biotechnology field, polypeptide of the present invention can be protected the nearly all Organ and tissue of body as the tissue protective polypeptide, especially for the Various Tissues inflammation, has good protection, treatment and healing effect.The present invention relates to the technology such as Solid-phase Polypeptide chemosynthesis, polypeptide large scale purification and freeze-drying, physical and chemical property determining, treatment application.
Background technology
Exogenous and endogenous damage factor can cause the various damaging pathologies of cell, meanwhile the reaction of series of complex then occurs in the part of body and whole body, with limitation and elimination damage factor, removing and absorption necrotic tissue, cell, and repair damage, the defensive reaction of Here it is body is called inflammation.Inflammation is very common and important basic pathology process, and most of common disease and the frequently-occurring disease (such as furuncle, carbuncle, pneumonia, hepatitis, ephritis etc.) of the trauma infection contamination of body surface and each organ all belong to diseases associated with inflammation.
Change into a series of local reactions at center with vascular system, be conducive to remove and eliminate virulence factor, oozing out of liquid can be diluted toxin, engulfs the carrying necrotic tissue and is beneficial to regeneration and reparation, makes virulence factor be confined to inflammation part and the unlikely whole body that spreads.Therefore, inflammation is the defensive reaction of body, is favourable to body usually, if there is not inflammatory reaction, people can not be full of in the physical environment of pro-inflammatory cytokine in this by long-term survival.But inflammation also has potential very large hazardness to body, and serious anaphylaxis can jeopardize patient's life; Pericardial cavity inner cellulose exudate machineization can form constrictive pericarditis, and then affects heart function; The inflammation that betides brain essence or meninx can cause that intracranial pressure raises, even the formation hernia cerebri causes the vital center pressurized and causes patient death; Vocal cords acute inflammation oedema can cause suffocating in addition, etc.So reaction that controls inflammation of under a stable condition, should taking measures.Based on above reason, the present invention has announced a kind of new polypeptide with organization protection's activity.
Multiple protein and the peptide that is used for the treatment of various humans and animals various disease arranged at present.These albumen or polypeptide have much be produce in vivo and can from animal or human body, extract for the preparation of pharmaceutical composition.Also having much is on the basis of these materials, and by chemosynthesis, chemical modification etc. technique means manually obtains, and is widely used in the various diseases treatment.Above-mentioned substance has: Regular Insulin, erythropoietin, BMP, Interferon, rabbit, Somatostatin, Sostatin, Leuprolide etc.Purpose of the present invention is announced a kind of preparation method with novel polypeptide of organization protection's activity, is used for the treatment of inflammation.Clinical experiment shows that polypeptide of the present invention for the Various Tissues inflammation, has good protection, treatment and healing effect.
Summary of the invention
The present invention relates to a kind of new polypeptide with organization protection's activity.This polypeptide contains 8-15 amino-acid residue, molecular weight is 900-1600 dalton and has the Various Tissues prolection.The present invention has announced its sequence, chemical synthesis process and the application in treatment thereof.Announced simultaneously and adopted method and the polypeptide of the present invention of polypeptide of the present invention and the various medicine types of one or more pharmaceutical carriers combination preparation to have good protection for various tissue inflammations, treatment and healing effect.Although the concrete mechanism of action is also indefinite, when polypeptide of the present invention gives very low dosage (being generally milligram or nanogram/kg body weight) in many ways nearly all Organ and tissue is demonstrated provide protection.Multinomial experimentation on animals shows, polypeptide of the present invention can weaken the damage of the tissues such as liver, lung, colon and stomach that caused by chemical induction, suppresses Inflammatory response, reduces digestive tract ulcer, reduces the damage of liver and biliary tract, also has anxiety and antidepressant effect.Can promote vasculogenesis, promote wound healing etc., can reverse unusual in the parkinsonism model of being induced by neurotoxin MPTP.In addition, have significant provide protection for stomach mucous membrane, can also the amelioration of inflammation reaction.
The peptide sequence that the present invention announces has following general structure:
【Xaa?Zaa?Pro?Pro?Pro?Xaa?Yaa?Pro?Ala?Asp?Zaa?Ala?Xaa?Xaa?Xaa】
Perhaps:
[Zaa?Pro?Pro?Pro?XaaYaa?Pro?Ala]
Wherein Xaa is neutral fat family amino-acid residue, particularly Ala, bAla, Leu, Ile, Gly, Val, Nle or Nva,
Yaa is alkaline amino acid residue, particularly Lys, Arg, Orn or His,
Zaa is acidic amino acid residue, particularly Glu, Asp, Aad or Apm.
The concrete combination of the aminoacid sequence of polypeptide of the present invention is recorded in the sequence table.
After polypeptide of the present invention is synthetic, the free state form can be made, also cationic salts can be made, as: tfa salt (trifluoroacetate), HCl salt (hydrochloride), acetate form.
Polypeptide of the present invention can be selected and one or more pharmaceutical carrier combinations, makes the multi-medicament formulation, is used for the treatment of.These pharmaceutical dosage forms are contained: formulation part or the whole body administrations such as injection solution, tablet, creme, capsule, ointment, lotion, tongue lozenge.Dosage is preferably 10
-5～10
-2In the scope of mg/kg body weight, with higher concentration whole body or the topical of 0.1%-0.5%.Determine it is well known to those skilled in the art for the optimal dose of concrete treatment.
Specific implementation method
Below in conjunction with concrete implementation method synthetic, the purifying of this polypeptide and physicochemical property and the treatment in the bovine mammary gland inflammation are further elaborated.
Embodiment 1: the chemosynthesis of polypeptide and purifying:
Take the aminoacid sequence shown in the SEQ ID No.2 as the example chemically synthesized polypeptide.
Adopt the Fmoc solid phase method synthetic:
Adopt the solid phase synthesis instrument, selecting the val-2 chlorine trityl resin of 0.5 gram is initial resin, adds successively in the building-up reactions pipe:
Fmoc-leu，Fmoc-gly，Fmoc-ala，Fmoc-Asp(otbu)fmoc-asp(otbu)，Fmoc-ala，Fmoc-pro，Fmoc-lys(boc)，FMoc-gly，FMoc-pro，Fmoc-pro，FMoc-pro，Fmoc-Glu(otbu)，Fmoc-gly.
Add DIC/DMF solution and HOBT/DMF solution and DMF solution in the reagent bottle.Start synthetic instrument and connect computer, the input synthesis program begins to synthesize.
Synthetic aftertreatment:
Resin is taken out from the building-up reactions post, put into the Glass Containers that the sintered glass filter disc is arranged at a bottom, use successively respectively DMF (dimethyl formamide), DCM (methylene dichloride), MeOH (methyl alcohol), DCM, MeOH5-10 resin volume flushing resin doubly.Resin lyophilize after the flushing.
The resin flushing is complete, and other gets a test tube with glass stopper, configures 20 milliliters of lytic reagents, and distributing style is as follows:
(1): add 17.5 milliliters of TFA (trifluoroacetic acid) (TFA has severe corrosive and volatility, should operate) in test tube in ventilating kitchen
(2): add the EDC0.5 milliliter
(3): add 1 milliliter of thioanisole
(4): add 0.4 milliliter of anisole
After above reagent all adds, slightly shake mixing, add the Glass Containers of putting into resin.Reacted 2.5 hours.Question response is complete, with way separation resin and the lytic reagent of vacuum filtration.Lytic reagent is put into a balloon flask, and the way pressure reducing and steaming liquid substance with rotary evaporation only stays solid.
Other gets 100 milliliters cold diethyl ether and adds in the balloon flask, and have a large amount of white precipitate materials this moment, is thick polypeptide.Centrifugation ether and polypeptide.Abandon the supernatant ether, polypeptide continues with the cold diethyl ether washing, and is centrifugal, obtains the polypeptide crude product.
The Freeze Drying Equipment freeze-drying of polypeptide crude product is used for next step purifying.
The purifying of synthetic product:
Polypeptide crude product 1 gram is dissolved in 5 milliliters the pure water, filters upper preparation scale high performance liquid chromatography.Chromatogram is joined the C18 reversed-phase column of 40*250mm.
Moving phase: A is 0.1% TFA+ water mutually
B is 0.1% TFA+ acetonitrile mutually.
Employing program: B rose to 55% from 5% in 40 minutes, collect main peak, obtained being dissolved in the polypeptide sterling of moving phase.
The polypeptide of mentioned solution state adopts the low-temperature rotary evaporation mode tentatively concentrated, then uses the Freeze Drying Equipment lyophilize.Obtain sterling.
Embodiment 2: stability test
Polypeptide of the present invention was tested its stability in 76 days and 120 days in 40 ℃ of insulations.Stability test adopts the HPLC method to measure.The concentration of polypeptide is 0.2% (w/v) in the aqueous solution: post, Kromasil100,5u, 250x 4.6mm; Moving phase, the water of 0.1% trifluoroacetic acid/second eyeball solution (0 to 50vol.%) is at 25 minutes inside gradient wash-outs, flow velocity 1ml/min; Detect: UV, 218nm.
In order to compare, use free polypeptide and Monoacetate thereof.
Table 1
Can show from table 1 data, polypeptide stability of the present invention is very high.Its solution still has the target substance more than 97% at 40 ℃ after 120 days.
Embodiment 3: the toxicity of polypeptide of the present invention
Adopt mouse stomach and injection system to measure the acute toxicity of polypeptide of the present invention.
Solution preparation:
Adopt 25 milligrams of polypeptide of the present invention, add aqua sterilisa to 20 milliliter, be configured to the solution that concentration is 1.25 mg/ml, for oral administration gavage.
Adopt 25 milligrams of polypeptide of the present invention, add aqua sterilisa to 10 milliliter, be configured to the solution that concentration is 2.5 mg/ml, for abdominal injection.
Dosage calculates:
Mouse oral administration gavage administration volume is 40 milliliters/kg body weight, therefore dosage is: 1.25mg/ml*40ml/kg=50mg/kg.
Mouse peritoneal drug administration by injection volume is 20 milliliters/kg body weight, therefore dosage is: 2.5mg/ml*20ml/kg=50mg/kg.
Get 80 of healthy mices, minute four groups of experiments.After the administration, at the 7th day and 14 days, observe, weigh.And after experiment finishes, weigh, put to death the anatomic observation internal.
Conclusion:
1, once-a-day oral administration gavage administration of mouse, 50 mg/kgs of body weight of dosage have no obvious acute toxic reaction.LD50＞50mg/kg。
2, mouse intraperitoneal injection once-a-day, 50 mg/kgs of body weight of dosage have no obvious acute toxic reaction.LD50＞50mg/kg。
From the above mentioned, polypeptide of the present invention has no obvious acute toxicity performance under 50 milligrams of per kilogram of body weight dosage.
Embodiment 4: contain the preparation of the tablet of polypeptide of the present invention
Embodiment 5: contain the preparation of the capsule of polypeptide of the present invention
Embodiment 6: contain the preparation of the solution of polypeptide
Embodiment 7: contain the preparation of the creme of polypeptide
Embodiment 8: to the therapeutic action of tissue inflammation.
Polypeptide of the present invention has good protection for the Various Tissues inflammation, treatment, and healing effect foreword:
With the suitable model that can represent the acute and/or chronic inflammatory diseases among the mankind, can be used for assessing the anti-inflammatory drug of present use.
Materials and methods:
At gastrointestinal damage (INDOMETHACIN (30mg/kg, subcutaneous), acetylsalicylic acid (400mg/kg, in the stomach) and diclofenac sodium (125mg/kg, intraperitoneal)) in the research, use regularly polypeptide of the present invention (10ug or 10ng/kg intraperitoneal), used in front 1 hour simultaneously and in medicine (INDOMETHACIN) application.In adjuvant arthritis (through tail administration 0.2ml freund's adjuvant) research (14 days, 30 days, 1 year), single-dose (using before or after the freund's adjuvant 1 hour) polypeptide of the present invention or use polypeptide of the present invention with scheme (0-the 14th day, 14-30 days and the 14th day-1 year) once a day.
The result:
With the NSAIA administration of research, polypeptide of the present invention has continued to reduce other the remarkable damages in the control rats stomach, and the damage in the INDOMETHACIN group Small Intestine.In adjuvant arthritis research, behind single-dose, even in the weaker rat for the treatment of with polypeptide of the present invention every day, the development of damage obviously reduces.As a kind of methods for the treatment of of established adjuvant arthritis, after only treating for 2 weeks, the beneficial effect of polypeptide just continues to occur, and more obvious rear 1 year of application.These data sheet understand that polypeptide is to anti-inflammatory and the provide protection of mucous membrane integral body.
Known in the treatment of adjuvant arthritis, relate to two kinds of different mechanism, the overreact of inflammation and delayed.Polypeptide of the present invention all has positive effect to the two.With respect to reference standard (being acetylsalicylic acid, INDOMETHACIN), the present invention also is effective at quite low dosage (ug and ng/kg are to mg/kg).In the application of this initial validity in every day in the adjuvant arthritis prevention even higher.Once or during the every day administration, higher dosage (10ug/kg) all is effectively, and than low dosage (10ng/kg), single administration is invalid, but when the every day administration, is effective.The unusual effect of polypeptide of the present invention in adjuvant arthritis (the preventing/treating effect, do not establish/adjuvant arthritis of establishing), can't observe with the medicine that uses at present.When using every day, glucocorticosteroid is effectively for the prevention of adjuvant arthritis, but is invalid in short.Immunosuppressor (high dosage) and non-steroid class anodyne are effective before treatment or after the treatment only respectively.
Therefore, based on the utmost point similarity between used model and the corresponding human disease, prove that the polypeptide of the present invention with mucous membrane protection characteristic can be used for treating the disease relevant with acute inflammation and chronic arthritis.In addition, polypeptide of the present invention can be used for treating the relevant disease of damage that the gastrointestinal damage that brings out with the delayed-type hypersensitivity disease with at the gi tract each several part is particularly brought out by medicine such as NSAIA.
Polypeptide of the present invention has obvious treatment provide protection except above-mentioned tissue is had the therapeutic action equally for damaged cell cell and organ.
Claims (4)
1. a peptide species is characterized in that, the aminoacid sequence of described polypeptide is selected from the polypeptide of the aminoacid sequence shown in the SEQ ID No.1-11.
2. the polypeptide of claim 1 record is characterized in that, contains 8-15 amino-acid residue, molecular weight is 900-1600 dalton and has the Various Tissues prolection.Described polypeptide has general structure: [Xaa Zaa Pro Pro Pro Xaa Yaa Pro Ala Asp Zaa Ala Xaa Xaa Xaa] or: [Zaa Pro Pro Pro Xaa Yaa Pro Ala],
Wherein Xaa is neutral fat family amino-acid residue, particularly Ala, bAla, Leu, Ile, Gly, Val, Nle or Nva,
Yaa is alkaline amino acid residue, particularly Lys, Arg, Orn or His,
Zaa is acidic amino acid residue, particularly Glu, Asp, Aad or Apm.
3. a pharmaceutical preparation is characterized in that, the polypeptide of claim 1 or 2 records is selected and one or more pharmaceutical carrier combinations, makes the multi-medicament formulation.These pharmaceutical dosage forms are contained: formulation part or the whole body administrations such as injection solution, tablet, creme, capsule, ointment, lotion, tongue lozenge.
4. the polypeptide of claim 1,2 or 3 records or pharmaceutical preparation are used for the treatment of tissue inflammation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102804884A CN103012555A (en) | 2011-09-20 | 2011-09-20 | Preparation method for new polypeptide having tissue protection activity, and application of new polypeptide in treatment |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102804884A CN103012555A (en) | 2011-09-20 | 2011-09-20 | Preparation method for new polypeptide having tissue protection activity, and application of new polypeptide in treatment |
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| CN103012555A true CN103012555A (en) | 2013-04-03 |
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| CN2011102804884A Pending CN103012555A (en) | 2011-09-20 | 2011-09-20 | Preparation method for new polypeptide having tissue protection activity, and application of new polypeptide in treatment |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104072588A (en) * | 2014-06-22 | 2014-10-01 | 马恒标 | Tissue-protection active polypeptides and application thereof |
| CN109481331A (en) * | 2018-11-12 | 2019-03-19 | 广州艾玫格生物科技有限公司 | A kind of body protective polypeptide and preparation method thereof with antiallergic effect |
-
2011
- 2011-09-20 CN CN2011102804884A patent/CN103012555A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104072588A (en) * | 2014-06-22 | 2014-10-01 | 马恒标 | Tissue-protection active polypeptides and application thereof |
| CN109481331A (en) * | 2018-11-12 | 2019-03-19 | 广州艾玫格生物科技有限公司 | A kind of body protective polypeptide and preparation method thereof with antiallergic effect |
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Application publication date: 20130403 |