CN108434459A - A kind of polypeptide drugs conjugate and its preparation method and application - Google Patents
A kind of polypeptide drugs conjugate and its preparation method and application Download PDFInfo
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
The present invention provides a kind of polypeptide drugs conjugate and its preparation method and application, the polypeptide drugs conjugate includes Ticagrelor and CREKA polypeptides, and the Ticagrelor is connect with CREKA polypeptides；Polypeptide drugs conjugate provided by the invention, by Ticagrelor by being connect with polypeptide, compared to existing platelet suppressant drug, it can either realize the high concentration enrichment at tumor tissues position, also it can be enriched in atherosclerotic tissue high concentration, it can be achieved with inhibiting the function of blood platelet without influencing platelet function during normal blood circulation in specific region in this way, the bleeding risk that conventional platelet suppressant drug is reduced while increasing drug availability, to realize that the metastases of better and safer inhibit or prevent the function of acute coronary artery syndrome.
Description
Technical field
The invention belongs to medicinal chemistry art, it is related to a kind of polypeptide drugs conjugate and its preparation method and application.
Background technology
Malignant tumour is current one of the principal disease for threatening human life and health, its infiltration and transfer is that its is pernicious
The characteristic mark of change.Tumour cell through blood transfer be tumour carry out far-end transfer main path, and at present clinically
Most of tumor patient treatment failures and dead principal element.
Metastases process includes that tumour cell passes through the vascular endothelial cells of tumor tissues and moves out entrance from original site
Blood circulation system, tumour cell are formed with blood operation and tumour cell in three key links of implantation of metastasis site,
Transfer process is related to the interaction between various kinds of cell adhesion molecule, extracellular matrix and other haemocytes.The study found that blood
Platelet plays vital effect during metastases, and specific mechanism of action includes mainly the following aspects：①
Blood platelet is activated into the tumour cell in blood circulation, the two assembles to form tumor bolt, to protect tumour cell from blood
The attack of turbulent flow and immune system；2. there is point that can mutually stick with tumour cell and vascular endothelial cell in platelet surface
Son makes it that can either stick and merge with the endothelial cell of damage, also it is swollen can to play promotion with tumor cell adhesion
The function served as bridge of oncocyte-endothelial cell adhesion, to help the vascular endothelial cell of tumour cell and metastasis site to stick；③
When tumour cell is after metastasis site is implanted into, blood platelet can directly facilitate tumour cell by secreting the multiple biological activities factor
Growth and breeding, and promote angiogenesis, provide suitable microenvironment for tumour growth.Wherein tumour cell and blood platelet is mutual
Effect forms cancer embolus (platelet aggregation that i.e. tumour cell induces, tumorcell-induced
Plateletaggregation, TCIPA) and its extracellular proteinase destroy capilary to enter surrounding tissue be tumour hematogenous metastasis
Rate-limiting step.
Therefore, inhibit the function of blood platelet that will be expected to become the powerful measure for inhibiting metastases.Have at present multiple anti-
Antiplatelet drug is employed successfully in the research for inhibiting Nasopharyngeal neoplasms on animal model.As blood platelet depleted mice is being tested
Property metastasis models in Lung metastases number significantly reduce；It can effectively inhibit blood platelet using GPIIbIIIa monoclonal antibodies
With the adherency of tumour cell, to inhibit metastases；Using platelet ADP receptor inhibitors Ticagrelor in mouse model
It successfully inhibits the transfer of tumour cell and extends survival time of mice.However, the inhibition or knockout by whole body system are dynamic
There are systemic bleeding risks for blood platelet in object, become limitation such methods and are applied to clinical key factor.
On the other hand, atherosclerosis (atherosclerosis) is the common pathological basis of cardiovascular and cerebrovascular disease,
And lead to the major reason of death.Pathological research show the occurrence and development of atherosclerosis include lipid infiltration,
Platelet activation, thrombosis, inner film injury, inflammatory reaction, oxidative stress, activation of vascular smooth muscle cells etc..Although many
Scholar is it is proposed that different theories about AS pathogenesis, but platelet activation is the committed step in normal coagulation mechanism,
It is also the major reason that vascular atherosclerosis disease such as acute coronary artery syndrome (ACS) pathologic thrombus is formed.Therefore, resist
Anti-platelet therapy is a very important aspect of Cardial or cerebral vascular diseases treatment.It is currently available that oral anti-diabetic agent object
Including aspirin, P2Y12 receptor antagonists clopidogrel and prasugrel, they can improve atherosclerotic's
Ischemia symptom.However, since current oral antidiabetic preparation has larger ischemic risk and higher hemorrhagic tendency, seek
Looking for new reducing, ischemic events occur and the medicine of hemorrhage risk is imperative.
Therefore, how the inhibition blood platelet of specificity is combined with tumour cell, and how to make platelet suppressant drug
Specificity is attached to atherosclerosis position, and does not influence blood platelet normal coagulation function, is both technical fields
Bottleneck problem.It is considered that the targeting Delivery of existing antiplatelet drug is realized, to realize at tumor tissues position or move
Pulse atherosclerosis site specific high concentration is enriched with, and is the key that solve the problems, such as this.
Polypeptide coupling drug (peptide drug conjugate, PDC) is a kind of novel coupling drug.It is to rely on
Targeting structural domain of the peptide chain of about 10 amino acid in one end as targets neoplastic cells, the other end is the drug for having biological function
Molecule realizes the efficient targeting Delivery of drug molecule, reaches the science purpose of oncotherapy Synergy and attenuation.With antibody coupling drug
(antibody drug conjugate, ADC) is compared, its molecular weight smaller is not easy to cause immune response；With antibody producing
Complex techniques process compare, PDC can be more efficient completely by being chemically synthesized, and is easier to make for purifying.Therefore, PDC
The development trend of next-generation targeted drug is increasingly becomed.Based on this, the present invention is directed to by tumor blood vessel targeted polypeptide
(CREKA) it is coupled to existing platelet suppressant drug and forms a kind of novel polypeptide drug conjugates together, be expected to realize efficiently low
The inhibition metastases of poison and the scientific goal for inhibiting progression of atherosclerosis.
Invention content
The purpose of the present invention is to provide a kind of polypeptide drugs conjugates and its preparation method and application.
To reach the invention purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of polypeptide drugs conjugate, the polypeptide drugs conjugate includes replacing card lattice
Thunder and CREKA polypeptides, the Ticagrelor are connect with CREKA polypeptides.
Polypeptide drugs conjugate provided by the invention is small compared to existing blood by Ticagrelor by being connect with polypeptide
Plate inhibitor can either realize the high concentration enrichment at tumor tissues position, also can be rich in atherosclerotic tissue high concentration
Collection can be achieved with inhibiting the function of blood platelet without influencing blood platelet work(during normal blood circulation in specific region in this way
Can, the bleeding risk of conventional platelet suppressant drug is reduced while increasing drug availability, to realize better and safer
Metastases inhibit or prevent acute coronary artery syndrome function.
Polypeptide drugs conjugate provided by the invention, is a kind of completely new drug conjugates, and Ticagrelor is common in individually
Medication, and be prepared into polypeptide molecule drug combination, then without any correlative study.
Ticagrelor is researched and developed by Astrazeneca AB of the U.S. and in the selective adp receptor-P2Y12 of listing in 2011 suppressions
Preparation belongs to new chemical classes-cyclopenta triazolo pyrimidine (CPTP) class, is a kind of novel platelet aggregation inhibitor, choosing
Selecting property Adenosine diphosphonic acid (ADP), the platelet activation for inhibiting ADP to mediate and aggregation, the mechanism of action with clopidogrel
It is similar.Unlike but, the interaction between Ticagrelor and blood platelet P2Y12 receptors has invertibity, to thrombus
Formation inhibited, transmitted without conformational change and signal, and the platelet function after drug withdrawal in blood is also fast therewith
Quick-recovery.Although it can preferably protect platelet counts and function, bleeding risk is still had in clinical application.
CREKA (Cys-Arg-Glu-Lys-Ala) polypeptide is the pentapeptide identified using internal peptide library selection, its energy and tumour
Blood coagulation plasma protein in blood vessel combines, and then is located in tumor vessel or in atherosclerotic blood vessel, is that drug is set
Widely applied target polypeptide during meter.Therefore, Ticagrelor is combined to form new polypeptide drugs coupling with CREKA polypeptides
Object, then can specificity the platelet function inhibited inside tumor tissues, or specificity inhibits atherosclerosis position
Platelet function, without influencing the platelet function during normal blood circulation, the inhibition to realize high-efficiency low-toxicity is swollen
Tumor metastasis or the ultimate aim for preventing acute coronary artery syndrome.
Preferably, the bridging agent includes straight chain fatty dicarboxylic anhydride.
Preferably, the straight chain fatty dicarboxylic anhydride include ethanedioic acid acid anhydride, malonic anhydride, succinic anhydride, glutaric anhydride, oneself two
Any one in acid anhydrides, pimelic acid acid anhydride, azelaic acid acid anhydride or sebacic anhydride.
Linking agent used in the present invention has good biocompatibility, has compared with similar functions nano-carrier, peace
Full property is more preferable.
Preferably, the CREKA polypeptides are the pentapeptide of Cys-Arg-Glu-Lys-Ala compositions.
Polypeptide used in the present invention has good biocompatibility, has compared with similar functions nano-carrier, safety
Property it is more preferable, while there is good target function, effect protrudes.
Preferably, the structure of polypeptide drugs conjugate of the present invention is specifically shown in formula I：
Wherein, R is straight chain fatty base.
Illustratively such as：When bridging agent is ethanedioic acid acid anhydride, R CH2-CH2；When bridging agent is malonic anhydride, R is
CH2-CH2-CH2。
In the present invention, Ticagrelor should neither influence the blood of Ticagrelor in such a way that bridging agent is connect with polypeptide
Platelet inhibits function, nor affects on the tumour and atherosclerosis target function of CREKA polypeptides.Preferably, replacing card lattice
The connection site of thunder is selected as free hydroxyl, and the connection site of CREKA polypeptides is selected as the free amino of N-terminal；And if other
Position is connected with bridging agent, then the activity of CREKA polypeptides is destroyed, from the function without polypeptide.
Second aspect, the present invention provides a kind of preparation method of polypeptide drugs conjugate as described in relation to the first aspect, institutes
Stating preparation method includes：CREKA polypeptides are synthesized with polypeptide solid-state reaction method, then Ticagrelor is connected on CREKA polypeptides
Obtain the polypeptide drugs conjugate.
Preferably, Ticagrelor is connected on CREKA polypeptides by using bridging agent.
Polypeptide drugs conjugate of the present invention is made by Solid-phase synthesis peptides method, and preparation method is simple, is easy to technique and puts
Greatly, the preparation process of monoclonal antibody has been compared, it is at low cost, it is efficient.
Preferably, the preparation method comprises the following steps：
(1) CREKA polypeptides are synthesized with polypeptide solid-state reaction method；
(2) product is obtained by the reaction in the presence of catalyst and alkaline reagent in CREKA polypeptides and bridging agent in solvent；
(3) polypeptide is obtained by the reaction with Ticagrelor in the presence of catalyst and alkaline reagent in the product that step (2) obtains
Drug conjugates crude product, cleaved, washing, dry, chromatogram purification obtain the polypeptide drugs conjugate.
Preferably, the resin used in solid-phase synthesis described in step (1) is 2- chlorine trityl chloride resins.
Preferably, the catalyst described in step (2) and step (3) is O- benzotriazole-tetramethylurea hexafluorophosphoric acid
Salt (HBTU), I-hydroxybenzotriazole (HOBT) and N, N- diisopropylcarbodiimide (DIC).
Preferably, the bridging agent, O- benzotriazole-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole and N,
The mass ratio of N- diisopropylcarbodiimide is (1-2):1:(1-2):(1-2), such as can be 1:1:1:1、1.5:1:1.4:
1.6、1.8:1:1.3:1.7 or 2:1:2:2.
Preferably, alkaline reagent described in step (2) and step (3) is n,N-diisopropylethylamine (DIEA).
Preferably, the mass ratio of the alkaline reagent and I-hydroxybenzotriazole is (1.5-3):1, such as can be 1.5:
1、1.8:1、2:1、2.4:1、2.5:1、2.8:1 or 3:1.
Preferably, solvent described in step (2) and step (3) is the mixed solvent that dimethylformamide and methanol form.
Preferably, it is cut into described in step (3) and is cut polypeptide from resin using cutting liquid.
Preferably, the cutting liquid be by mass percentage by 94.5% trifluoroacetic acid (TFA), 2% water,
The mixed solution that 2.5% 1,2 dithioglycols (EDT) and 1% tri isopropyl silane (TIS) are formed.
Preferably, the washing is to be washed using ether.
As optimal technical scheme, preparation method provided by the invention specifically includes following steps：
(1) resin swelling：Weigh the 2- chlorine trityl chloride resins (2-Chlorotrityl that degree of substitution is 0.5mmol/g
Chloride Resin resins), resin is put into reaction tube, add methylene chloride (DCM), oscillation.
(2) first amino acid is connect：Solvent is leached out by husky core, Fmoc-Ala-OH is added, is added after adding DIEA
A small amount of DCM dissolvings, oscillation.Then directly add methanol reaction, it is therefore an objective to which end socket finally uses dimethylformamide (DMF) and DCM to hand over
For cleaning 6 times.
(3) it is deprotected：20% Piperidine/DMF solution is added to react.
(4) it detects：Piperidine solution is taken out, resin is taken, is washed three times with ethyl alcohol, ninhydrin, KCN, phenol solution each one is added
Drop, heating, change navy blue is positive reaction.
(5) it washs：It is washed twice using DMF successively, methanol is washed twice, and DMF is washed twice.
(6) it is condensed：Fmoc-Lys (Boc)-OH, HBTU, DIEA, HOBT, DIC is weighed, with DMF dissolvings are lacked as possible, is added
Enter reaction tube to be reacted.
(7) it washs：Successively twice with DMF, methanol is washed twice, and DMF is washed twice.
(8) operation of three to seven steps, the amino acid being sequentially connected from right to left in sequence are repeated.
(9) bridging agent is connected：Bridging agent is weighed, HBTU, DIEA, HOBT, DIC are added anti-with DMF dissolvings are lacked as possible
Then Ying Guan is washed twice with DMF, methanol is washed twice, and DMF is washed twice, filtering and washing 6 times.
(10) Ticagrelor is connect：Weigh 0.1g Ticagrelors, HBTU, DIEA, HOBT, DIC, with as possible less DMF
Dissolving is added reaction tube, is then washed twice with DMF, methanol is washed twice, and DMF is washed twice, filtering and washing 6 times.
(11) it washs：It is washed three times using methanol.
(12) polypeptide is cut from resin：Prepare cutting liquid：TFA 94.5%；Water 2.5%；EDT 2.5%；TIS 1%.
Resin is fitted into flask or centrifuge tube, resin and cutting liquid proportional are according to 10mL/g, isothermal vibration.
(13) drying washing：Lysate is dried up as possible with nitrogen, is separated out with ether layer, then six times are washed with ether, so
Room temperature volatilizes afterwards.Up to polypeptide drugs conjugate crude product.
(14) crude product polypeptide drugs conjugate is taken to carry out chromatogram purification, chromatographic condition is：Mobile phase is water and acetonitrile, time
High performance liquid chromatography (HPLC) start gradient is first balanced 5min then sample introductions, start gradient water by 30min, gradient elution
95%, acetonitrile 5% terminates ratio water 5%, and acetonitrile 95% determines that target product goes out peak position.
(15) it prepares：The sample that will have been dissolved does sample introduction preparation.Preparation HPLC balance 10min, start gradient water 95%,
Acetonitrile 5% terminates gradient water 25%, acetonitrile 75%, gradient timetable 40min.The sample come out from detector is collected, it finally will be pure
Solution freeze-drying after change, had both obtained polypeptide drugs conjugate, powdered polypeptide had packed, -20 DEG C of preservations.
The third aspect is preparing antineoplastic the present invention provides a kind of polypeptide drugs conjugate as described in relation to the first aspect
Object prepares the application prevented in acute coronary artery syndrome drug.
Polypeptide drugs conjugate provided by the invention passes through special at tumor tissues position or atherosclerosis position
Property inhibition blood platelet function, inhibit tumor tissues far-end transfers or prevent acute coronary artery syndrome to control to realize
Therapeutic effect.
Compared with the existing technology, the invention has the advantages that：
Polypeptide drugs conjugate provided by the invention is small compared to existing blood by Ticagrelor by being connect with polypeptide
Plate inhibitor can either realize the high concentration enrichment at tumor tissues position, also can be rich in atherosclerotic tissue high concentration
Collection can be achieved with inhibiting the function of blood platelet without influencing blood platelet work(during normal blood circulation in specific region in this way
Can, the bleeding risk of conventional platelet suppressant drug is reduced while increasing drug availability, to realize better and safer
Metastases inhibit or prevent acute coronary artery syndrome function.
Description of the drawings
Fig. 1 is the synthetic method flow diagram of polypeptide drugs conjugate in the embodiment of the present invention 1.
Fig. 2 is polypeptide drugs conjugate HPLC purification result figures in the embodiment of the present invention 2.
Fig. 3 is polypeptide drugs conjugate Mass Spectrometer Method result figure in the embodiment of the present invention 2.
Fig. 4 is polypeptide drugs conjugate platelet aggregation inhibitory activity result curve figure in the embodiment of the present invention 3.
Fig. 5 be in the embodiment of the present invention 4 polypeptide drugs conjugate to combine coagulation thrombus albumen laser confocal microscope
Observe result figure.
Fig. 6 is that polypeptide drugs conjugate inhibits metastases comparative result figure in the embodiment of the present invention 5.
Fig. 7 is that polypeptide drugs conjugate targets atherosclerotic plaque fluorescence results figure in the embodiment of the present invention 6.
Specific implementation mode
The technical solution further illustrated the present invention below by specific implementation mode.Those skilled in the art should be bright
, the embodiment, which is only to aid in, understands the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
In the present embodiment, it is prepared by the following method polypeptide drugs conjugate, specifically includes following steps：
(1) the 2- chlorine trityl chloride resin 0.3g that degree of substitution is 0.5mmol/g are weighed, resin is put into reaction tube,
Add DCM (15mL/g), vibrates 10min.
(2) resin solution obtained by step (1) is leached out into solvent by husky core, the Fmoc-Ala-OH of 0.037g is added, then
A small amount of DCM dissolvings are added after the DIEA of 0.08g is added, vibrate 2h.Then the purpose of direct plus methanol (about 1mL) reaction 15min, is
End socket finally uses DMF and DCM alternately cleaning 6 times.
(3) 20% Piperidine/DMF solutions of 15ml (15mL/g) are added to react the resin for connecting Ala amino acid in step (2)
20min。
(4) by step (3), treated, and resin takes out piperidine solution, takes more than ten grainy resins, is washed three times with ethyl alcohol, indenes is added
Triketone, KCN, each drop of phenol solution, 105 DEG C of -110 DEG C of heating 5min, change navy blue is positive reaction.
(5) after determining that step (4) reaction is positive, step (3) resin is washed twice with DMF (10mL/g), first
Alcohol (10mL/g) washes twice, and DMF (10mL/g) is washed twice.
(6) 0.210g Fmoc-Lys (Boc)-OH, HBTU 0.04g, DIEA 0.08g, HOBT 0.04g, DIC are weighed
0.04g is added the reaction tube of step (5), reacts 40min with DMF dissolvings are lacked as possible.
(7) step (6) reacted resin being washed twice with DMF (10mL/g), methanol (10mL/g) washes twice,
DMF (10mL/g) is washed twice.
(8) operation of three to seven steps, the amino acid being sequentially connected from right to left in sequence are repeated.
(9) succinic anhydride 0.05g, HBTU 0.04g, DIEA 0.08g, HOBT 0.04g, DIC 0.04g is weighed, is used
Lack DMF dissolvings as possible, step (8) reaction tube is added.React 40min.Then it is washed twice with DMF (10mL/g), methanol
(10mL/g) is washed twice, DMF (10mL/g) filtering and washing 6 times twice.
(10) 0.1g Ticagrelors are weighed, HBTU 0.04g, DIEA 0.08g, HOBT 0.04g, DIC 0.04g are used
Lack DMF dissolvings as possible, step (9) reaction tube is added.React 40min.Then DM (10mL/g) is washed twice, methanol (10mL/g)
It washes twice, DMF (10mL/g) filtering and washing 6 times twice.
(11) resin after step (10) processing is washed three times with methanol (10mL/g).
(12) cutting liquid (10/g) is prepared：TFA 94.5%；Water 2%；EDT 2.5%；TIS 1%.Step (11) are set
Fat is fitted into flask or centrifuge tube, and resin and cutting liquid proportional are according to 10mL/g, isothermal vibration, cleavage reaction 120min.
(13) lysate in step (12) is dried up as possible with nitrogen, is separated out with ether layer, then six times are washed with ether, so
Room temperature volatilizes afterwards.Up to polypeptide drugs conjugate crude product.
The flow diagram of synthesis step is as shown in Figure 1.
Embodiment 2
In the present embodiment, purified polypeptide drug conjugates by the following method, specifically include following steps：
(1) crude product polypeptide drugs conjugate is taken to be put into vessel.It is dissolved with the acetonitrile solution of 2-5mL 50%.
(2) by 0.45 μm of membrane filtration of step (1) solution.
(3) it analyzes：3 μ L are taken to analyze crude product with analysis level HPLC.Mobile phase is water and acetonitrile, time 30min, and gradient is washed
It is de-, HPLC start gradients are first balanced into 5min then sample introductions, start gradient water 95%, acetonitrile 5% terminates ratio water 5%, second
Nitrile 95% determines that target product goes out peak position.
(4) it prepares：The sample that will have been dissolved does sample introduction preparation.It prepares HPLC and balances 10min, start gradient water 95%, second
Nitrile 5% terminates gradient water 25%, acetonitrile 75%, gradient timetable 40min.The sample come out from detector is collected, purity is obtained
Purpose product more than 90%.
Fig. 2 is the schematic diagram of HPLC purification results, and product is analyzed by mass spectrometry, as shown in Figure 3.Complex chart 2 and Fig. 3
It can obtain, polypeptide drugs conjugate synthesizes successfully, and successfully purifies.
Embodiment 3
In the present embodiment, the antiplatelet for polypeptide drugs conjugate being investigated using the method for influencing platelet aggregation rate is lived
Property, method is as follows：
(1) the polypeptide drugs conjugate 1mg prepared is weighed, the drug solution that 1mL ethyl alcohol is prepared into 1mg/mL is added；Root
The Ticagrelor dosage that equivalent concentration is calculated according to conjugate and Ticagrelor Molecular weights, weighs Ticagrelor powder 0.42mg,
1mL ethyl alcohol is added and fully dissolves.
(2) by step (1) conjugate and Ticagrelor solution according to 1:2 ratio doubling dilutions 6 times, i.e., it is corresponding
Conjugate drug concentration be 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL,
0.015625mg/mL, it is spare.
(3) porcine vein 20ml is taken, with 3.8% sodium citrate 9:1 anti-freezing is collected in plastic centrifuge tube.By blood with
Anti-coagulants mixes well.10min is centrifuged with 200g at room temperature, upper layer ecru suspension is sucked out up to platelet rich plasma
(PRP), remaining that 10min is centrifuged with 2000g, supernatant is taken, platelet poor plasma (PPP) is obtained.
(4) BornShi turbidimetry principles are pressed and uses MPG-3E type Multifunctional two-ways road blood pool instrument.Take PRP200 μ L in
In opacity tube, the 10 μ L of liquid or PBS of various concentration are separately added into, 37 DEG C of incubation 3min in preheating gate.Then it is stirred in bar magnet
It mixes down and is separately added into derivant ADP (60 μm of ol/L), returned to zero with PPP and detect maximum platelet aggregation rate in 5min.
Test results are shown in figure 4, such as Fig. 4 results it is found that polypeptide drugs conjugate has similar resist with Ticagrelor
Biologically active pdgf.
Embodiment 4
In the present embodiment, the target function of the coagulation thrombus albumen of polypeptide drugs conjugate is investigated, method is as follows：
(1) the conjugate 0.5mg prepared is weighed, 0.5mL PBS are added, fully dissolve.
(2) the amine-modified rhodamine 1mg of maleimide is weighed, 1mL PBS are added and draw 0.5mL fully after dissolving
It is added in the solution of step (1) preparation, is protected from light reaction overnight.
(3) 5mL porcine veins are taken, with 3.8% sodium citrate 9:1 anti-freezing is collected in plastic centrifuge tube.3000g is centrifuged
10min takes supernatant.
(4) it takes step (3) to centrifuge supernatant 1mL, the CaCl of 0.4M is added2100 μ of fibrin ferment of solution 100 μ L, 0.1U/mL
L, suction are applied to after beating mixing on glass slide, and 4 DEG C are incubated overnight.
(5) reaction solution and each 200 μ L of rhodamine solution that prepared by aspiration step (2), are evenly coated in step (4)
On the glass slide prepared, it is protected from light and is incubated 10min.
(6) glass slide after using PBS solution rinsing step (5) to be incubated 6 times.
(7) covered observes the rhodamine fluorescing matter on two groups of fragmentations under laser confocal microscope.
The results are shown in Figure 5, and the target it is found that the coagulation thrombus albumen of polypeptide drugs conjugate is compared by Fig. 5 and control group
It is good to function.
Embodiment 5
In the present embodiment, the inhibition metastases ability of polypeptide drugs conjugate is investigated, method is as follows：
(1) the polypeptide drugs conjugate for preparing embodiment 1 enters nude mice metastatic breast cancer 4T1 by tail vein injection
It in model, is administered once within every 2 days, 3 experimental groups of setting, i.e. physiological saline group, Ticagrelor group, polypeptide drugs conjugate group is even
It is 10mg/kg to join object and the effective dose of Ticagrelor group.
The results are shown in Figure 6, it will be appreciated from fig. 6 that compared with physiological saline group, Ticagrelor group can be effective with conjugate group
Inhibit the spontaneous lung transfer of 4T1 tumours, but the inhibition efficiency of conjugate group will be apparently higher than Ticagrelor medicine group, say
The polypeptide drugs conjugate of the bright present invention can greatly improve the anti-tumor metastasis effect of original drug.
Embodiment 6
In the present embodiment, the atherosclerotic plaque for investigating polypeptide drugs conjugate targets ability, and method is as follows：
(1) by way of High-fat diet Apo E knock out mice, atherosclerosis animal model is prepared；
(2) the amine-modified FITC dyestuff 1mg of maleimide are weighed, 1mL PBS are added, fully after dissolving, 0.5mL is drawn and adds
Enter into the polypeptide drugs conjugate solution of 1mg/mL, is protected from light reaction overnight.
(3) solution that step (2) prepares is entered into the mouse that modeling is completed by tail vein injection, and dyestuff control is set
Group after 3h is administered, puts to death animal and corresponding tissue is taken to be imaged.
The results are shown in Figure 7, and as shown in Figure 7, compared with dyestuff control group, the polypeptide drugs conjugate group of dye marker exists
There are more fluorescence signals in the aorta of mouse, show that polypeptide drugs conjugate is targeted with good atherosclerosis
Effect.
Applicant states that the present invention illustrates polypeptide drugs conjugate and its preparation side of the present invention by above-described embodiment
Method and application, but the invention is not limited in above-mentioned processing steps, that is, do not mean that the present invention has to rely on above-mentioned processing step
It could implement.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to raw material selected by the present invention
Equivalence replacement and the addition of auxiliary element, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope
Within.
Claims (10)
1. a kind of polypeptide drugs conjugate, which is characterized in that the polypeptide drugs conjugate includes Ticagrelor and CREKA more
Peptide, the Ticagrelor are connect with CREKA polypeptides.
2. polypeptide drugs conjugate according to claim 1, which is characterized in that the Ticagrelor by bridging agent with
CREKA polypeptides connect；
Preferably, the bridging agent includes straight chain fatty dicarboxylic anhydride；
Preferably, the straight chain fatty dicarboxylic anhydride includes ethanedioic acid acid anhydride, malonic anhydride, succinic anhydride, glutaric anhydride, adipic acid
Any one in acid anhydride, pimelic acid acid anhydride, azelaic acid acid anhydride or sebacic anhydride.
3. polypeptide drugs conjugate according to claim 1 or 2, which is characterized in that the CREKA polypeptides are Cys-Arg-
The pentapeptide of Glu-Lys-Ala compositions.
4. polypeptide drugs conjugate according to any one of claim 1-3, which is characterized in that the polypeptide drugs coupling
The structure of object is shown in formula I：
Wherein, R is straight chain fatty base.
5. the preparation method of the polypeptide drugs conjugate according to any one of claim 1-4, which is characterized in that the system
Preparation Method includes：CREKA polypeptides are synthesized with polypeptide solid-state reaction method, then Ticagrelor is connected on CREKA polypeptides and is obtained
The polypeptide drugs conjugate；
Preferably, Ticagrelor is connected on CREKA polypeptides by using bridging agent.
6. preparation method according to claim 5, which is characterized in that the preparation method comprises the following steps：
(1) CREKA polypeptides are synthesized with polypeptide solid-state reaction method；
(2) product is obtained by the reaction in the presence of catalyst and alkaline reagent in CREKA polypeptides and bridging agent in solvent；
(3) polypeptide drugs are obtained by the reaction with Ticagrelor in the presence of catalyst and alkaline reagent in the product that step (2) obtains
Conjugate crude product, cleaved, washing, dry, chromatogram purification obtain the polypeptide drugs conjugate.
7. preparation method according to claim 6, which is characterized in that the tree used in solid-phase synthesis described in step (1)
Fat is 2- chlorine trityl chloride resins.
8. the preparation method described according to claim 6 or 7, which is characterized in that the catalysis described in step (2) and step (3)
Agent is O- benzotriazole-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole and N, N- diisopropylcarbodiimide；
Preferably, the bridging agent, O- benzotriazole-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole and N, N- bis-
The mass ratio of diisopropylcarbodiimide is (1-2):1:(1-2):(1-2)；
Preferably, alkaline reagent described in step (2) and step (3) is n,N-diisopropylethylamine；
Preferably, the mass ratio of the alkaline reagent and I-hydroxybenzotriazole is (1.5-3):1；
Preferably, solvent described in step (2) and step (3) is the mixed solvent that dimethylformamide and methanol form.
9. according to the preparation method described in any one of claim 6-8, which is characterized in that being cut into described in step (3) makes
Polypeptide is cut from resin with cutting liquid；
Preferably, the cutting liquid is by mass percentage by 94.5% trifluoroacetic acid, 2% water, the 1 of 2.5%, 2 second
Two mercaptan and 1% the mixed solution that is formed of tri isopropyl silane；
Preferably, the washing is to be washed using ether.
10. the polypeptide drugs conjugate according to any one of claim 1-4 is preparing antitumor drug or is preparing prevention
Application in acute coronary artery syndrome drug.
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