CN101045746A - Anti-thrombocyte tetratitanate-NB06 and its application - Google Patents
Anti-thrombocyte tetratitanate-NB06 and its application Download PDFInfo
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- CN101045746A CN101045746A CN 200710087476 CN200710087476A CN101045746A CN 101045746 A CN101045746 A CN 101045746A CN 200710087476 CN200710087476 CN 200710087476 CN 200710087476 A CN200710087476 A CN 200710087476A CN 101045746 A CN101045746 A CN 101045746A
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Abstract
This invention relates to a new thrombocyte anti-aggregation tetrapeptide - NB06, its amino acid sequence is omega - aminocaprylic acid - glycine - aspartic acid - tryptophyl amine peptide. The amidated carboxyl terminal could enlarge top space of 'V' type peptide, thereby enhance biology activity of object compound, along with it advance its ability of resisting hydrolization, establish good foundation for its using in preparing drugs for thrombocyte anti-aggregation and cerebrovascular disease treatment.
Description
Technical field:
The present invention relates to a kind of new linear tetrapeptide compound in medical biotechnology field with antiplatelet aggregative activity.
Background technology:
Thrombosis, metastases and destruction of bone absorb, and are having a strong impact on human beings'health for a long time.In recent years, along with the theory of cells involved interphase interaction---cell adhesion theory constantly perfect, the adhesion between cell and iuntercellular, cell and extracellular matrix, transhipment are considered to the key link that above disease takes place.Further discover, the combination of a kind of tripeptide sequence RGD (Arg-Gly-Asp) and respective ligand thereof, be the essential link of cell interaction, block the research of this link, will have important theory and realistic meaning the pathogeny and the clinical treatment of above disease.
RGD sequence on the Fibrinogen α chain combines with platelet glycoprotein (GP) IIb/IIIa acceptor generation specificity, is the final common pathway that platelet thrombus forms.Just because of the RGD sequence on the Fibrinogen can combine with thrombocyte generation specificity, make the RGD of synthetic and analogue also might suppress Fibrinogen competitively and combine with hematoblastic.So blocking platelet is assembled in theory fully, suppresses the thrombosis that any reason causes.Thereby, become a focus of the many drugmakers in world research based on the new drug research of RGD and analogue thereof.
Though the RGD sequence on the Fibrinogen is being brought into play important effect in vivo, as medicine, only natural RGD sequence can not reach proper intensity and selectivity.Because the correct space conformation that it has broken away from native protein to be had.So RGD in natural protein conformation and near aminoacid sequence, very important for recognition process.Aminoacid sequence on every side can make RGD keep correct conformation, and particularly with RGD sequence next-door neighbour's amino-acid residue, correct conformation is more meaningful for keeping.
To discovering of RGD class peptide structure activity relationship, the three-dimensional conformation of RGD class peptide is " V " font, and the size of " V " word angle, the i.e. distance of " V " font top guanidine radicals and carboxyl, closely related with the biological activity of peptide, distance is big can be better and receptors bind.The 4th amino acids that links to each other with the RGD sequence is bigger to its activity influence.Tomiyama etc. are by evidence, and the carboxyl terminal of RGD class peptide is good with hydrophobic amino acid, and hydrophobicity is strong more, and it suppresses the ability strong more [J.Biol Chem, 1992] of Fibrinogen and thrombocyte GPIIb/IIIa receptors bind.
It is sad that the inventor once transform the N-terminal arginine of RGD class peptide as omega-amino-, and the 4th amino acids is adopted the amino acid of high hydrophobicity, obtain serial highly active RGD class peptide, successfully applied for and obtained Chinese invention patent (ZL 02123924.X).
In order further to improve the biologic activity and the anti-hydrolysis ability thereof of target compound, become anti-platelet aggregation medicine of new generation with better function in the hope of exploitation, the inventor is on the foregoing invention basis, carboxyl terminal to the tetrapeptide compound is transformed, through experimental study, finally selected the C end structure of tryptophyl amine, and then a kind of new tetrapeptide be provided as RGD class peptide---omega-amino-is sad-glycine-aspartic acid-tryptophyl amine peptide (NB06).New tetrapeptide array structure involved in the present invention is not seen relevant report so far as yet.
Summary of the invention:
The invention provides a kind of new tetrapeptide, its aminoacid sequence is: omega-amino-is sad-and glycine-aspartic acid-tryptophyl amine peptide.Amidated C-terminal makes the vertical distance of " V " font peptide increase, and has strengthened the biologic activity of target compound, has also improved its anti-hydrolysis ability simultaneously.
The invention provides the method for the said antiplatelet tetrapeptide compound-NB06 of a kind of preparation.This method is based on the disclosed aminoacid sequence X-Gly-Asp-Y of inventor's aforementioned patent, selects for use omega-amino-sad wherein X, and Y selects for use tryptophyl amine to realize.The main innovate point of NB06 of the present invention is the selection to Y.In aforementioned patent of invention, stipulate: " Y is any one among Trp, Leu, Val, Ile, Phe, the Ser ", being natural amino acid, end is a carboxyl.Y corresponding among the present invention has selected tryptophyl amine for use, and end has carried out the amidation modification, does not belong to any one that defines in the aforementioned patent.
The present invention provides the pharmaceutical composition of new tetrapeptide compound-NB06 again, and said composition is to be effective constituent with new tetrapeptide compound-NB06, and contains pharmaceutically acceptable one or more carriers.
The present invention also provides said new tetrapeptide compound-NB06 platelet aggregation inhibitory activity experiment, stability experiment, the result shows, compare with the disclosed compd A oGDW of aforementioned patent of invention peptide, the platelet aggregation inhibitory activity of NB06 of the present invention has improved nearly four times, and has better biological stability.
The compound of structure of the present invention is as increasing by one or several natural or alpha-non-natural amino acids, the new compound that can form at the N of NB06 end or C end.
The compound of structure of the present invention is as carrying out conventional chemically modified, the new compound that also can form to NB06.
The invention advantage:
Advantage of the present invention and positively effect are, through modification and transformation to aminoacid sequence X-Gly-Asp-Y, especially selected for use tryptophyl amine to replace Y, end has been carried out the amidation modification, thereby improved the platelet aggregation inhibitory activity and the stability of the said new tetrapeptide-NB06 compound of the present invention greatly, established good basis for developing clinical effective anti-platelet aggregation medicine of new generation.
Description of drawings:
The high pressure liquid chromatography (HPLC) of Fig. 1-NB06 compound;
The mass spectrometric detection analysis of Fig. 2-NB06 compound.
Embodiment:
Provide some embodiment below, purpose only is to help to understand the present invention better, rather than limitation of the present invention.
＜embodiment 1 〉: the solid phase synthesis of NB06
Take by weighing 76mg Rink Amide mbha resin (carrying capacity is the 0.55mM/ gram), use the methylene dichloride swelling after 1 hour in the polypeptide reaction flask, suction filtration is removed methylene dichloride; Add 5 milliliters of 20% pyridines/dimethyl formamide, room temperature vibration mixing 10 minutes is to slough the Fmoe protecting group; Suction filtration is removed deprotection liquid, repeats deprotection once; Take by weighing the tryptophane of 5 times of excessive Fmoc protections, be dissolved among the 2M DIEA 1ml (or 0.5ml), add excessive 5 times 0.5M HBTU/HoBT, mixing, room temperature activation 10 minutes; Protect amino acid to be transferred to reactor activatory, the vibration mixing, room temperature reaction is after 60 minutes, and negative pressure is taken out liquid; Add methylene dichloride 4ml, vibration mixing 30 seconds, negative pressure is taken out washings, repeated washing totally 7 times; The peptide resin that takes a morsel detects condensation efficiency with ninhydrin method, and reaction negative explanation condensation is complete, can carry out next step, otherwise repeats condensation once; Repeat deprotection-condensation reaction, respectively with the sad introducing peptide chain of aspartic acid, glycine and omega-amino-.Reaction vacuumizes drying with peptide-resin after finishing.
＜embodiment 2 〉: the cutting of solid phase synthesis of peptide-resin
Get peptide-resin that embodiment 1 obtains, add 2 milliliters of lysates (95% trifluoracetic acid, 2.5% water, 2.5% tri isopropyl silane), stirring reaction is two hours under the room temperature; Suction filtration goes out to cut liquid afterwards, and with the resin in a small amount of TFA washing reaction bottle, suction filtration goes out TFA and integrates with cutting liquid; Negative pressure is drained the cutting liquid that leaches, row ice ether sedimentation, and precipitation separation is used the 1ml water dissolution.
＜embodiment 3 〉: the purifying of NB06
The dissolved cleaved products that takes a morsel is carried out high pressure liquid chromatography (HPLC), determines the retention time of product, and separated product.The condition of anti-phase high-pressure liquid phase purifying is: C8 post (3.9 * 150mm, waters company), mobile phase A: 0.1%TFA/H
2O, B:0.1%TFA/ acetonitrile, gradient elution, flow velocity 1ml/mm, monitoring 214nm absorption peak.Result such as Fig. 1: two main elution peaks are arranged, and are respectively 17.422 minutes elution peak (being designated as P1) and 17.712 minutes elution peak (being designated as P2); Wherein P1 is the purpose compound, and its peak area accounts for 40% of the total area.Collect the P1 elution peak,, be the NB06 behind the purifying through vacuum-freeze-dry.
＜embodiment 4 〉: the mass spectrum of NB06 (MS) is identified
Carry out mass spectrometric detection in the Chinese Academy of Medical Sciences synthetic chamber of medicine.Result such as Fig. 2: main peak is obvious, and molecular weight is 516.2, conforms to theoretical molecular, and assorted peak is low and lack.Mass spectrometric detection shows that synthetic NB06 molecular weight is correct, the purity height.
＜embodiment 5 〉: the active contrast experiment of NB06 and AoGDW peptide (omega-amino-sad-glycine-aspartic acid-tryptophane)
1) the external anti human platelet aggregation experimental technique of RGD class peptide
Adopt human blood through ulnar vein on an empty stomach early morning, with 3.8% Sodium Citrate anti-freezing (volume ratio of blood and antithrombotics is 9: 1), under the room temperature, 1000r/ minute centrifugal 8 minutes, get upper plasma and be platelet rich plasma (PRP, platelet-rich plasma), separate PRP.Yu Xuezai with 3000r/ minute centrifugal 10 minutes, is got upper plasma and gets platelet poor plasma (PPP, platelet-poor plasma), adjust PRP, make platelet count be about 250,000/μ L with PPP.Get PRP400 μ L, the RGD class peptide 50ul that adds 0.9%NaCl (negative control) and different concns respectively, adopt turbidimetry for Determination platelet aggregation rate (Helena Packs-4 type platelet aggregation instrument), 37 ℃ with 900r/ minute, on platelet aggregation instrument, stir, hatch and add adenosine diphosphate (ADP) (ADP) 50ul after 2 minutes, begin simultaneously to measure.Minute is 10 minutes.Write down maximum platelet aggregation rate.
The calculating of anticoagulant rate:
A0: the maximum platelet aggregation rate of physiological saline group
The maximum platelet aggregation rate of A:RGD class peptide
2) experimental result of the external anti human platelet aggregation of AoGDW (n=6)
| Concentration (uM) | Aggregation rate (%) | Inhibiting rate (%) |
| 0 0.5 1 2 4 8 | 88.3±4.56 82.6±4.13 72.6±3.82 52.4±3.01 27.3±1.62 11.1±1.17 | 0 6.4±2.53 17.8±2.98 40.7±3.26 69.7±3.57 87.4±1.41 |
By last table data, trace the inhibition curve, to choose linearity range and carry out regression analysis, its equation of linear regression is:
Y＝8.3429X+3.6 (R
2＝0.9836)
By regression equation calculation, the IC of the external anti human platelet aggregation of AoGDW
50Value is 5.56uM.
3) experimental result of the external anti human platelet aggregation of NB06 (n=6)
| Concentration (uM) | Aggregation rate (%) | Inhibiting rate (%) |
| 0 0.5 1 2 4 8 | 88.3±4.56 78.2±4.47 56.2±4.04 28.4±2.94 8.6±1.31 4.5±0.86 | 0 11.4±2.07 36.3±2.74 67.8±3.91 90.3±2.15 94.9±1.03 |
By last table data, trace the inhibition curve, to choose linearity range and carry out regression analysis, its equation of linear regression is:
Y＝31.5X+4.8 (R
2＝0.985)
By regression equation calculation, the IC of the external anti human platelet aggregation of NB06
50Value is 1.43uM.
4) the active contrast experiment of NB06 and AoGDW peptide
AoGDW is that activity is a kind of preferably in the described RGD class of the patent of invention 02123924.X peptide, and structure and NB06 are approaching.Experimental result according to embodiment 1, the activity of the anti human platelet aggregation of NB06 is 3.89 times of AoGDW, be new tetrapeptide-omega-amino-provided by the invention sad-glycine-aspartic acid-tryptophyl amine peptide (NB06), its activity has improved about 4 times.
＜embodiment 6 〉: the active stability experiment of NB06 anti human platelet aggregation
According to embodiment 1 described activity determination method, carry out the determination of activity of NB06 anti human platelet aggregation.For observing intensity action time of medicine, i.e. stability in blood plasma, after respectively NB06, AoGDW and RGDS (arginine-glycine-aspartic acid acid-Serine) (final concentration be respectively 1,4 and 20uM) being hatched 3h with 37 ℃ of PRP (add-on is the same), with concentration is that 20uM ADP induced platelet is assembled, and measures maximum platelet aggregation rate.Minute is 10 minutes.Used instrument is a Helena Packs-4 type platelet aggregation instrument.The same terms is divided into control group and is done directly to detect, and hatches and the active difference of not hatching with contrast.Simultaneously, make negative control, to measure maximum platelet aggregation rate with physiological saline.Establish 3 parallel laboratory tests for every group, measure maximum platelet aggregation rate after, calculate gathering inhibiting rate (method of calculation reference example 5) of each group.After each inhibiting rate averaged, contrast the activity change of different medicine anticoagulant.
Experimental result is as follows:
1) the physiological saline group maximum platelet aggregation rate of not hatching is 87.6, and the platelet aggregation rate of hatching after 3 hours is 87.1;
2) the stability experiment result of NB06 (final concentration is 1uM)
The platelet aggregation rate of not hatching group is respectively: 56.2,55.8,54.3, and corresponding gathering inhibiting rate is: 35.8%, 36.3%, 38.0%, on average assembling inhibiting rate is 36.7%.The platelet aggregation rate of hatching the 3h group is respectively: 55.1,55.7,54.8, and corresponding gathering inhibiting rate is: 36.7%, 36.1%, 37.1%, on average assembling inhibiting rate is 36.6%.
Contrast is hatched group and is not hatched group, and the anticoagulant rate of NB06 is indifference almost.This explanation, in platelet rich plasma (PRP), hatch 3 hours after, NB06 have and hatch before identical platelet aggregation inhibitory activity, promptly NB06 has fabulous stability.
3) the stability experiment result of AoGDW (final concentration is 4uM)
The platelet aggregation rate of not hatching group is respectively: 25.8,27.4,24.5, and corresponding gathering inhibiting rate is: 70.6%, 68.7%, 72.0%, on average assembling inhibiting rate is 70.4%.The platelet aggregation rate of hatching the 3h group is respectively: 38.3,32.6,40.7, and corresponding gathering inhibiting rate is: 56.0%, 62.6%, 53.3%, on average assembling inhibiting rate is 57.3%.
Contrast is hatched group and is not hatched group, and the anticoagulant rate of AoGDW has than obvious variation.Compare with not hatching group, AoGDW is hatched platelet aggregation inhibitory activity after 3 hours and is dropped to 81.4% before hatching.This explanation AoGDW has degraded to a certain degree in platelet rich plasma, cause the decline of its platelet aggregation inhibitory activity.
4) the stability experiment result of RGDS (final concentration is 20uM)
The platelet aggregation rate of not hatching group is respectively: 68.5,70.2,71.6, and corresponding gathering inhibiting rate is: 21.8%, 19.9%, 18.3%, on average assembling inhibiting rate is 20.0%.The platelet aggregation rate of hatching 3 hours groups is respectively: 85.9,84.2,87.0, and corresponding gathering inhibiting rate is: 1.4%, 3.3%, 0.1%, on average assembling inhibiting rate is 1.6%.
Contrast is hatched group and is not hatched group, and the anticoagulant rate of RGDS has very obvious variation.Compare with not hatching group, the platelet aggregation inhibitory activity that RGDS is hatched behind the 3h almost disappears, only for hatching preceding active 8%.This explanation RGDS has tangible degraded in platelet rich plasma, cause its platelet aggregation inhibitory activity obviously to descend, even disappear.
5) the stability experiment result of three kinds of RGD class peptides contrast
By above-mentioned experimental result as can be seen, with blood plasma external hatch 3 hours jointly after, NB06 still has almost 100% activity; The activity of AoGDW is reduced to about 80%; And the activity of RGDS is almost completely lost.This explanation, NB06 has good antiplatelet stability, and this C-terminal structure amidated with it has confidential relation.
Claims (4)
1, a kind of new antiplatelet tetrapeptide compound-NB06, its aminoacid sequence be omega-amino-sad-glycine-aspartic acid-tryptophyl amine.
2, the application of the described tetrapeptide compound-NB06 of claim 1 in preparation anti-platelet aggregation and treatment cardiovascular and cerebrovascular diseases medicament.
3, the pharmaceutical composition of the described compound of claim 1 is characterized in that it is an activeconstituents with the above-claimed cpd of contained treatment significant quantity, and contains one or more pharmaceutically acceptable carriers.
4, as the application of composition as described in the claim 3 in preparation anti-platelet aggregation and treatment cardiovascular and cerebrovascular diseases medicament.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112125953A (en) * | 2020-07-23 | 2020-12-25 | 昆明医科大学第一附属医院 | Polypeptide for resisting platelet aggregation |
| CN113429457A (en) * | 2021-06-16 | 2021-09-24 | 昆明医科大学第一附属医院 | Polypeptide for resisting platelet aggregation and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112125953A (en) * | 2020-07-23 | 2020-12-25 | 昆明医科大学第一附属医院 | Polypeptide for resisting platelet aggregation |
| CN112125953B (en) * | 2020-07-23 | 2022-09-16 | 昆明医科大学第一附属医院 | Polypeptide for resisting platelet aggregation |
| CN113429457A (en) * | 2021-06-16 | 2021-09-24 | 昆明医科大学第一附属医院 | Polypeptide for resisting platelet aggregation and application thereof |
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