CN108434459B - Polypeptide drug conjugate and preparation method and application thereof - Google Patents
Polypeptide drug conjugate and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- 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
- A61K47/62—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 the modifying agent being a protein, peptide or polyamino acid
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
The invention provides a polypeptide drug conjugate and a preparation method and application thereof, wherein the polypeptide drug conjugate comprises ticagrelor and CREKA polypeptide, and the ticagrelor is connected with the CREKA polypeptide; compared with the existing platelet inhibitor, the polypeptide drug conjugate provided by the invention has the advantages that the ticagrelor is connected with the polypeptide, so that the high-concentration enrichment of tumor tissue parts can be realized, the high-concentration enrichment of atherosclerotic tissues can also be realized, the function of inhibiting platelets in a specific area can be realized without influencing the platelet function in the normal blood circulation process, the drug availability is increased, the bleeding risk of the conventional platelet inhibitor is reduced, and the function of better and safer tumor metastasis inhibition or acute coronary syndrome prevention is realized.
Description
Technical Field
The invention belongs to the field of medicinal chemistry, and relates to a polypeptide drug conjugate, and a preparation method and application thereof.
Background
Malignant tumors are one of the major diseases threatening human life and health at present, and the infiltration and metastasis thereof are characteristic markers of the malignancy thereof. The blood metastasis of tumor cells is the main way for the distant metastasis of tumor, and is also the main factor of treatment failure and death of most tumor patients clinically at present.
The tumor metastasis process comprises three main links, namely the migration of tumor cells from a primary part to a blood circulation system after the tumor cells penetrate through vascular endothelial cells of tumor tissues, the running of the tumor cells along with blood and the implantation of the tumor cells at a metastasis part, and relates to the interaction among various cell adhesion molecules, extracellular matrix and other blood cells. The research finds that the blood platelet plays a crucial role in the process of tumor metastasis, and the specific action mechanism mainly comprises the following aspects: firstly, the tumor cells entering the blood circulation activate platelets, and the platelets are aggregated to form tumor emboli, so that the tumor cells are protected from the attack of blood turbulence and an immune system; the surface of the platelet has molecules which can be mutually adhered with tumor cells and vascular endothelial cells, so that the platelet can be adhered and fused with damaged endothelial cells and can also be adhered with the tumor cells, and the platelet has a bridge function of promoting the adhesion of the tumor cells and the endothelial cells, thereby helping the adhesion of the tumor cells and the vascular endothelial cells at a metastatic part; and thirdly, after the tumor cells are implanted in the metastasis part, the platelets can directly promote the growth and the propagation of the tumor cells by secreting various bioactive factors, promote angiogenesis and provide a proper microenvironment for the growth of the tumor. The tumor cells and the platelets interact to form cancer emboli (i.e. tumor cell-induced platelet aggregation, TCIPA) and the secretory protease thereof to destroy the microvasculature to enter the surrounding tissues are the rate-limiting step of tumor hematogenous metastasis.
Therefore, inhibition of platelet function would be a promising means for inhibiting tumor metastasis. At present, a plurality of antiplatelet drugs are successfully used for the research of inhibiting the tumor cell metastasis on animal models. For example, the number of lung metastases in platelet-deficient mice in experimental tumor metastasis models is significantly reduced; the GPIIbIIIa monoclonal antibody can effectively inhibit the adhesion of platelets and tumor cells, thereby inhibiting tumor metastasis; the platelet ADP receptor inhibitor ticagrelor successfully inhibits the metastasis of tumor cells in a mouse model and prolongs the survival period of mice. However, systemic suppression or knockout of platelets in animals presents a systemic hemorrhagic risk and is an important factor limiting the clinical applications of such methods.
On the other hand, atherosclerosis (atherosclerosis) is a common pathological basis for cardiovascular and cerebrovascular diseases and is also a significant cause of death of patients. Pathological studies have shown that the development of atherosclerosis includes lipid infiltration, platelet activation, thrombosis, intimal damage, inflammatory response, oxidative stress, vascular smooth muscle cell activation, and the like. Although many scholars have put forth different theories about the pathogenesis of AS, platelet activation is a key step in the normal coagulation mechanism and is also a major cause of pathological thrombosis in vascular atherosclerotic diseases such AS Acute Coronary Syndrome (ACS). Therefore, antiplatelet therapy is a very important aspect of the treatment of cardiovascular and cerebrovascular diseases. Currently available oral antiplatelet drugs include aspirin, clopidogrel, the P2Y12 receptor antagonist, and prasugrel, all of which improve the ischemic symptoms in patients with atherosclerosis. However, since current oral antiplatelet agents have a greater risk of ischemic events and a higher tendency to bleed, it is imperative to find new therapeutic agents that reduce the risk of ischemic events and bleeding.
Therefore, how to specifically inhibit the platelet from binding to tumor cells and how to make the platelet inhibitor specifically bind to the atherosclerosis site without affecting the normal blood coagulation function of the platelet is a bottleneck problem in the two technical fields. The key to solving the problem is that the targeted delivery of the existing antiplatelet drugs is realized, so that the specific high-concentration enrichment at the tumor tissue site or the atherosclerosis site is realized.
Polypeptide conjugate (PDC) is a novel conjugate. The peptide chain with about 10 amino acids at one end is used as a targeting structural domain of a targeting tumor cell, and the other end is a drug molecule with a biological function, so that the high-efficiency targeting delivery of the drug molecule is realized, and the scientific aims of synergy and attenuation of tumor treatment are fulfilled. Compared with Antibody Drug Conjugate (ADC), the molecular weight of the antibody drug conjugate is smaller, and immune response is not easy to cause; compared to the complex process of antibody production, PDC can be synthesized entirely by chemical methods, with higher efficiency, and easier purification. Thus, PDC has become an increasingly developing trend of next-generation targeted drugs. Based on the technical scheme, the invention aims to couple the tumor blood vessel targeting polypeptide (CREKA) and the existing platelet inhibitor together to form a novel polypeptide drug conjugate, and is expected to realize the scientific targets of inhibiting tumor metastasis and inhibiting the development of atherosclerosis with high efficiency and low toxicity.
Disclosure of Invention
The invention aims to provide a polypeptide drug conjugate and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a polypeptide drug conjugate, which comprises ticagrelor and CREKA polypeptide, wherein the ticagrelor is connected with the CREKA polypeptide.
Compared with the existing platelet inhibitor, the polypeptide drug conjugate provided by the invention has the advantages that the ticagrelor is connected with the polypeptide, so that the high-concentration enrichment of tumor tissue parts can be realized, the high-concentration enrichment of atherosclerotic tissues can also be realized, the function of inhibiting platelets in a specific area can be realized without influencing the platelet function in the normal blood circulation process, the drug availability is increased, the bleeding risk of the conventional platelet inhibitor is reduced, and the function of better and safer tumor metastasis inhibition or acute coronary syndrome prevention is realized.
The polypeptide drug conjugate provided by the invention is a brand-new drug conjugate, and ticagrelor is commonly used in single drugs, but is prepared into a polypeptide molecule combined drug, so no related research is available.
Ticagrelor is a selective ADP receptor-P2Y 12 inhibitor developed by American Aspirin company and marketed in 2011, belongs to a new chemical class, namely cyclopentyl triazolopyrimidine (CPTP), is a novel platelet aggregation inhibitor, selectively antagonizes Adenosine Diphosphate (ADP), inhibits ADP-mediated platelet activation and aggregation, and has a similar action mechanism with clopidogrel. However, in contrast, the interaction between ticagrelor and the platelet P2Y12 receptor is reversible, inhibiting thrombus formation, without conformational changes and signaling, and with rapid recovery of platelet function in the blood following withdrawal. Although it can better preserve platelet number and function, there is still a bleeding risk in clinical applications. CREKA (Cys-Arg-Glu-Lys-Ala) polypeptide is a pentapeptide screened and identified by an in-vivo peptide library, can be combined with blood coagulation plasma protein in tumor blood vessels, is further positioned in the tumor blood vessels or in atherosclerotic blood vessels, and is a targeting polypeptide widely applied in the process of medicament design. Therefore, the ticagrelor and the CREKA polypeptide are combined to form a novel polypeptide drug conjugate, so that the platelet function in a tumor tissue or the platelet function in an atherosclerosis part can be specifically inhibited, the platelet function in a normal blood circulation process is not influenced, and the ultimate goal of inhibiting tumor metastasis or preventing acute coronary syndrome with high efficiency and low toxicity is realized.
Preferably, the linking agent comprises a linear fatty dianhydride.
Preferably, the linear aliphatic dianhydride comprises any one of oxalic anhydride, malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, azelaic anhydride or sebacic anhydride.
The linker used in the invention has good biocompatibility and better safety compared with a nano carrier with similar function.
Preferably, the CREKA polypeptide is a pentapeptide consisting of Cys-Arg-Glu-Lys-Ala.
The polypeptide used in the invention has good biocompatibility, better safety compared with a nano carrier with similar function, good targeting function and outstanding effect.
Preferably, the structure of the polypeptide drug conjugate of the present invention is specifically shown in formula I:
wherein R is a linear aliphatic group.
Exemplary are as follows: when the linking agent is oxalic anhydride, R is CH2-CH2(ii) a When the linking agent is malonic anhydride, R is CH2-CH2-CH2。
In the invention, the ticagrelor is connected with the polypeptide by the connecting agent, so that the platelet inhibition function of the ticagrelor and the tumor and atherosclerosis targeting function of the CREKA polypeptide are not influenced. Preferably, the attachment site of ticagrelor is selected as a free hydroxyl group, and the attachment site of CREKA polypeptide is selected as a free amino group at the N terminal; whereas if attached elsewhere to the linker, the CREKA polypeptide activity is disrupted and the polypeptide does not function.
In a second aspect, the present invention provides a method for preparing a polypeptide drug conjugate as described in the first aspect, the method comprising: and (3) synthesizing the CREKA polypeptide by a polypeptide solid-phase synthesis method, and then connecting ticagrelor to the CREKA polypeptide to obtain the polypeptide drug conjugate.
Preferably, ticagrelor is attached to the CREKA polypeptide by using a linker.
The polypeptide drug conjugate is prepared by a polypeptide solid phase synthesis method, the preparation method is simple, the process amplification is easy, and compared with the preparation process of a monoclonal antibody, the preparation method has the advantages of low cost and high efficiency.
Preferably, the preparation method comprises the following steps:
(1) synthesizing CREKA polypeptide by a polypeptide solid phase synthesis method;
(2) reacting CREKA polypeptide with a linking agent in a solvent in the presence of a catalyst and an alkaline reagent to obtain a product;
(3) and (3) reacting the product obtained in the step (2) with ticagrelor in the presence of a catalyst and an alkaline reagent to obtain a crude polypeptide drug conjugate, and cutting, washing, drying and carrying out chromatographic purification to obtain the polypeptide drug conjugate.
Preferably, the resin used in the solid phase synthesis method in step (1) is a 2-chlorotrityl chloride resin.
Preferably, the catalyst in the step (2) and the step (3) is O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBT) and N, N-Diisopropylcarbodiimide (DIC).
Preferably, the mass ratio of the connecting agent, the O-benzotriazole-tetramethylurea hexafluorophosphate, the 1-hydroxybenzotriazole and the N, N-diisopropylcarbodiimide is (1-2) to (1-2), and 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, for example.
Preferably, the basic agent in step (2) and step (3) is N, N-Diisopropylethylamine (DIEA).
Preferably, the mass ratio of the basic agent to 1-hydroxybenzotriazole is (1.5-3: 1, and may be, for example, 1.5:1, 1.8:1, 2:1, 2.4:1, 2.5:1, 2.8:1 or 3:1.
Preferably, the solvent in step (2) and step (3) is a mixed solvent of dimethylformamide and methanol.
Preferably, the cleavage in step (3) is cleavage of the polypeptide from the resin using a cleavage solution.
Preferably, the cutting fluid is a mixed solution consisting of 94.5% of trifluoroacetic acid (TFA), 2% of water, 2.5% of 1,2 Ethanedithiol (EDT) and 1% of Triisopropylsilane (TIS) in percentage by mass.
Preferably, the washing is with diethyl ether.
As a preferred technical scheme, the preparation method provided by the invention specifically comprises the following steps:
(1) swelling resin: a2-chlorotrityl chloride Resin (2-chlorotrityl chloride Resin) having a substitution degree of 0.5mmol/g was weighed out, and the Resin was put into a reaction tube, and Dichloromethane (DCM) was added thereto and shaken.
(2) Grafting with the first amino acid: the solvent was filtered off by suction through a sand core, Fmoc-Ala-OH was added, DIEA was added and a small amount of DCM was added to dissolve, and the mixture was shaken. Then methanol was added directly for reaction to cap and finally washed 6 times with Dimethylformamide (DMF) and DCM alternately.
(3) Deprotection: adding 20% piperidine DMF solution for reaction.
(4) And (3) detection: the piperidine solution is pumped out, the resin is taken out, washed with ethanol for three times, added with ninhydrin, KCN and phenol solution respectively in a drop manner, heated, and turned dark blue to be a positive reaction.
(5) Washing: two washes with DMF, two washes with methanol, and two washes with DMF were used in that order.
(6) Condensation: weighing Fmoc-Lys (Boc) -OH, HBTU, DIEA, HOBT and DIC, dissolving the materials with DMF as little as possible, and adding the materials into a reaction tube for reaction.
(7) Washing: two times with DMF, two times with methanol and two times with DMF in sequence.
(8) Repeating the three to seven steps, and connecting the amino acids in the sequence from right to left.
(9) Connecting a connecting agent: weighing the connecting agent, HBTU, DIEA, HOBT and DIC, dissolving the connecting agent, HBTU, DIEA, HOBT and DIC in DMF as little as possible, adding the mixture into a reaction tube, washing the mixture twice with DMF, washing the mixture twice with methanol, washing the mixture twice with DMF, and performing suction filtration and washing 6 times.
(10) Connecting ticagrelor: 0.1g of ticagrelor, HBTU, DIEA, HOBT and DIC are weighed, dissolved in DMF as little as possible, added into a reaction tube, washed twice with DMF, washed twice with methanol and washed twice with DMF, and filtered and washed 6 times.
(11) Washing: three washes with methanol were used.
(12) Cleavage of the polypeptide from the resin: preparing cutting fluid: 94.5% of TFA; 2.5 percent of water; 2.5 percent of EDT; and (3) TIS 1%. And (3) putting the resin into a flask or a centrifuge tube, shaking the resin and the cutting fluid at constant temperature according to the proportion of 10 mL/g.
(13) Drying and washing: drying the lysate with nitrogen, separating with diethyl ether, washing with diethyl ether for six times, and volatilizing at room temperature. Thus obtaining the crude product of the polypeptide drug conjugate.
(14) And (3) taking the crude polypeptide drug conjugate for chromatographic purification, wherein the chromatographic conditions are as follows: the mobile phase is water and acetonitrile, the time is 30min, gradient elution is carried out, High Performance Liquid Chromatography (HPLC) is firstly balanced for 5min by using an initial gradient and then sample injection is carried out, the initial gradient is 95 percent of water, the acetonitrile is 5 percent, the end proportion is 5 percent of water, the acetonitrile is 95 percent, and the peak position of the target product is determined.
(15) Preparation: and (5) preparing a sample injection for the dissolved sample. Preparative HPLC equilibration for 10min, initial gradient water 95%, acetonitrile 5%, end gradient water 25%, acetonitrile 75%, gradient time 40 min. Collecting sample from detector, lyophilizing purified solution to obtain polypeptide drug conjugate, sealing and packaging powdered polypeptide, and storing at-20 deg.C.
In a third aspect, the invention provides an application of the polypeptide drug conjugate in preparation of an anti-tumor drug or a drug for preventing acute coronary syndrome.
The polypeptide drug conjugate provided by the invention realizes the treatment effect of inhibiting the distal metastasis of tumor tissues or preventing acute coronary syndromes by the function of specifically inhibiting platelets at tumor tissue sites or atherosclerosis sites.
Compared with the prior art, the invention has the following beneficial effects:
compared with the existing platelet inhibitor, the polypeptide drug conjugate provided by the invention has the advantages that the ticagrelor is connected with the polypeptide, so that the high-concentration enrichment of tumor tissue parts can be realized, the high-concentration enrichment of atherosclerotic tissues can also be realized, the function of inhibiting platelets in a specific area can be realized without influencing the platelet function in the normal blood circulation process, the drug availability is increased, the bleeding risk of the conventional platelet inhibitor is reduced, and the function of better and safer tumor metastasis inhibition or acute coronary syndrome prevention is realized.
Drawings
FIG. 1 is a schematic flow chart of the synthesis method of the polypeptide drug conjugate in example 1 of the present invention.
FIG. 2 is a diagram showing the HPLC purification results of the polypeptide drug conjugate in example 2 of the present invention.
FIG. 3 is a diagram showing the results of mass spectrometric detection of the polypeptide drug conjugate in example 2 of the present invention.
Fig. 4 is a graph showing the result of the anti-platelet aggregation activity of the polypeptide drug conjugate in example 3 of the present invention.
FIG. 5 is a diagram showing the observation result of the polypeptide drug conjugate in example 4 of the present invention on a thromboxane conjugate by confocal laser microscopy.
FIG. 6 is a comparison of the results of the polypeptide drug conjugates of example 5 of the present invention in inhibiting tumor metastasis.
FIG. 7 is a graph showing the fluorescence results of the polypeptide drug conjugate targeting atherosclerotic plaques in example 6 of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In this example, the polypeptide drug conjugate was prepared by the following method, specifically including the following steps:
(1) 0.3g of 2-chlorotrityl chloride resin having a degree of substitution of 0.5mmol/g was weighed out, and the resin was put into a reaction tube, DCM (15mL/g) was added, and the mixture was shaken for 10 min.
(2) And (2) filtering the resin solution obtained in the step (1) by a sand core to remove the solvent, adding 0.037g of Fmoc-Ala-OH, adding 0.08g of DIEA, adding a small amount of DCM for dissolving, and oscillating for 2 h. Methanol (ca. 1mL) was then added directly to react for 15min to cap and finally washed 6 times with DMF and DCM alternately.
(3) The resin with the Ala amino acid attached in step (2) was reacted with 15mL of 20% piperidine DMF solution (15mL/g) for 20 min.
(4) And (4) pumping out the piperidine solution from the resin treated in the step (3), taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and a phenol solution into the resins respectively in a drop manner, heating the resins at the temperature of between 105 and 110 ℃ for 5min, and turning dark blue to be a positive reaction.
(5) After determining that the reaction of step (4) was positive, the resin of step (3) was washed twice with DMF (10mL/g), twice with methanol (10mL/g) and twice with DMF (10 mL/g).
(6) 0.210g of Fmoc-Lys (Boc) -OH, 0.04g of HBTU, 0.08g of DIEA, 0.04g of HOBT and 0.04g of DIC were weighed, dissolved in DMF as little as possible, and added to the reaction tube of step (5) to react for 40 min.
(7) The reacted resin from step (6) was washed twice with DMF (10mL/g), twice with methanol (10mL/g) and twice with DMF (10 mL/g).
(8) Repeating the three to seven steps, and connecting the amino acids in the sequence from right to left.
(9) 0.05g of succinic anhydride, 0.04g of HBTU, 0.08g of DIEA, 0.04g of HOBT and 0.04g of DIC are weighed, dissolved in DMF as little as possible and added into the reaction tube in the step (8). The reaction was carried out for 40 min. Then washed twice with DMF (10mL/g), twice with methanol (10mL/g) and twice with DMF (10mL/g) with suction filtration 6 times.
(10) 0.1g of ticagrelor, 0.04g of HBTU, 0.08g of DIEA, 0.04g of HOBT and 0.04g of DIC are weighed, dissolved in DMF as little as possible and added into the reaction tube in the step (9). The reaction was carried out for 40 min. Then, the mixture was washed twice with DM (10mL/g), twice with methanol (10mL/g) and twice with DMF (10mL/g) with suction filtration 6 times.
(11) The resin treated in step (10) was washed three times with methanol (10 mL/g).
(12) Preparing cutting fluid (10/g): 94.5% of TFA; 2% of water; 2.5 percent of EDT; and (3) TIS 1%. And (3) putting the resin in the step (11) into a flask or a centrifuge tube, shaking the resin and the cutting fluid at a constant temperature according to the proportion of 10mL/g, and carrying out a cutting reaction for 120 min.
(13) And (3) blowing the cracking liquid obtained in the step (12) to the greatest extent by using nitrogen, carrying out chromatography by using diethyl ether, washing for six times by using the diethyl ether, and then volatilizing at normal temperature. Thus obtaining the crude product of the polypeptide drug conjugate.
The scheme of the synthesis step is shown in FIG. 1.
Example 2
In this example, the polypeptide drug conjugate was purified by the following method, specifically including the following steps:
(1) and putting the crude polypeptide drug conjugate into a vessel. Dissolved with 2-5mL of 50% acetonitrile in water.
(2) The solution of step (1) was filtered through a 0.45 μm filter.
(3) And (3) analysis: 3 μ L of crude product was analyzed by analytical grade HPLC. And (3) carrying out gradient elution for 30min by using water and acetonitrile as mobile phases, balancing HPLC by using an initial gradient for 5min, then injecting a sample, and determining the peak position of the target product by using 95% of initial gradient water, 5% of acetonitrile, 5% of end proportion water and 95% of acetonitrile.
(4) Preparation: and (5) preparing a sample injection for the dissolved sample. Preparative HPLC equilibration for 10min, initial gradient water 95%, acetonitrile 5%, end gradient water 25%, acetonitrile 75%, gradient time 40 min. And collecting a sample from the detector to obtain the target product with the purity of more than 90%.
FIG. 2 is a schematic representation of the results of HPLC purification and mass spectrometry analysis of the product, as shown in FIG. 3. The combination of fig. 2 and fig. 3 shows that the polypeptide drug conjugate is successfully synthesized and purified.
Example 3
In this example, the antiplatelet activity of the polypeptide drug conjugate was examined using a method affecting the platelet aggregation rate as follows:
(1) weighing 1mg of the prepared polypeptide drug conjugate, and adding 1mL of ethanol to prepare a 1mg/mL drug solution; calculating the using amount of the ticagrelor with equivalent concentration according to the molecular weight of the conjugate and the ticagrelor, weighing 0.42mg of ticagrelor powder, adding 1mL of ethanol, and fully dissolving.
(2) And (2) diluting the conjugate in the step (1) and the ticagrelor solution for 6 times according to a 1:2 ratio multiple ratio, namely, the corresponding drug concentrations of the conjugate are 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL and 0.015625mg/mL for standby.
(3) 20ml of pig venous blood is collected in a plastic centrifuge tube with 3.8% sodium citrate 9:1 anticoagulation. And fully and uniformly mixing the blood and the anticoagulant. Centrifuging at room temperature for 10min at 200g, sucking out the upper layer beige suspension to obtain Platelet Rich Plasma (PRP), centrifuging at 2000g for 10min, and collecting the supernatant to obtain Platelet Poor Plasma (PPP).
(4) The MPG-3E type multifunctional double-channel blood coagulation instrument is adopted according to the principle of the Born's turbidimetry. mu.L of PRP was put in a turbidimetric tube, 10. mu.L of each drug solution or PBS was added thereto, and incubated in a pre-heated well at 37 ℃ for 3 min. Then adding an inducer ADP (60 mu mol/L) under the stirring of a magnetic bar, and detecting the maximum aggregation rate of the platelets within 5min by PPP zero setting.
The test result is shown in fig. 4, and as can be seen from the result in fig. 4, the polypeptide drug conjugate and ticagrelor have similar antiplatelet activity.
Example 4
In this example, the thrombin-targeting function of the polypeptide drug conjugate was examined as follows:
(1) 0.5mg of the prepared conjugate was weighed, and 0.5mL of PBS was added and sufficiently dissolved.
(2) Weighing 1mg of maleimide modified rhodamine dye, adding 1mL of PBS, fully dissolving, sucking 0.5mL of the dye, adding the dye into the solution prepared in the step (1), and keeping away from light for overnight reaction.
(3) 5mL of pig venous blood was collected in a plastic centrifuge tube with 3.8% sodium citrate 9:1 anticoagulation. Centrifuging at 3000g for 10min, and collecting supernatant.
(4) Taking 1mL of the centrifugal supernatant obtained in the step (3), and adding 0.4M CaCl2The solution 100U L, 0.1U/mL thrombin 100U L, suction and mixing, coated on glass slide, 4 degrees overnight incubation.
(5) And (3) sucking 200 mu L of each of the reaction solution prepared in the step (2) and the rhodamine dye solution, uniformly coating the reaction solution and the rhodamine dye solution on the glass slide prepared in the step (4), and incubating for 10min in a dark place.
(6) The slides incubated in step (5) were washed 6 times with PBS solution.
(7) And covering a cover glass, and observing the rhodamine fluorescence condition on the two groups of fragments under a laser confocal microscope.
As shown in fig. 5, the thrombin-targeting function of the polypeptide drug conjugate was found to be excellent in comparison with the control group in fig. 5.
Example 5
In this example, the ability of polypeptide drug conjugates to inhibit tumor metastasis was examined as follows:
(1) the polypeptide drug conjugate prepared in example 1 was injected into a nude mouse metastatic breast cancer 4T1 model via tail vein, 1 administration was performed every 2 days, 3 experimental groups were set, i.e., a physiological saline group, a ticagrelor group, and a polypeptide drug conjugate group, and the effective doses of the conjugate and the ticagrelor group were all 10 mg/kg.
As shown in fig. 6, as can be seen from fig. 6, compared with the normal saline group, both the ticagrelor group and the conjugate group can effectively inhibit spontaneous pulmonary metastasis of 4T1 tumor, but the inhibition efficiency of the conjugate group is significantly higher than that of the ticagrelor drug group, which indicates that the polypeptide drug conjugate of the present invention can greatly improve the anti-tumor metastasis effect of the original drug.
Example 6
In this example, the atherosclerotic plaque targeting ability of polypeptide drug conjugates was examined as follows:
(1) preparing an atherosclerosis animal model by feeding Apo E gene knockout mice with high-fat diet;
(2) weighing 1mg of maleimide-modified FITC dye, adding 1mL of PBS, fully dissolving, sucking 0.5mL of the dye, adding the dye into 1mg/mL of polypeptide drug conjugate solution, and keeping out of the sun overnight for reaction.
(3) Injecting the solution prepared in the step (2) into a mouse which is subjected to molding through tail vein, setting a dye control group, and killing the animal to take corresponding tissues for imaging after 3 hours of administration.
The result is shown in fig. 7, and it can be seen from fig. 7 that the dye-labeled polypeptide drug conjugate group has more fluorescence signals in the aorta of mice compared with the dye control group, indicating that the polypeptide drug conjugate has a good atherosclerosis targeting effect.
The applicant states that the polypeptide drug conjugate and the preparation method and application thereof are illustrated by the above examples, but the invention is not limited to the above process steps, i.e. the invention does not mean that the invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (13)
1. A polypeptide drug conjugate is characterized by comprising ticagrelor and CREKA polypeptide, wherein the ticagrelor is connected with the CREKA polypeptide through a connecting agent; the connecting agent is succinic anhydride, glutaric anhydride or adipic anhydride;
the CREKA polypeptide is pentapeptide consisting of Cys-Arg-Glu-Lys-Ala.
2. The polypeptide drug conjugate of claim 1, wherein the structure of the polypeptide drug conjugate is represented by formula I:
wherein R is C2-C4 straight-chain aliphatic group.
3. The method for preparing a polypeptide-drug conjugate according to claim 1 or 2, comprising: synthesizing a CREKA polypeptide by a polypeptide solid phase synthesis method, and then connecting ticagrelor to the CREKA polypeptide to obtain the polypeptide drug conjugate;
ticagrelor is attached to the CREKA polypeptide by using a linker which is succinic, glutaric or adipic anhydride.
4. The method of manufacturing according to claim 3, comprising the steps of:
(1) synthesizing CREKA polypeptide by a polypeptide solid phase synthesis method;
(2) reacting CREKA polypeptide with a linking agent in a solvent in the presence of a catalyst and an alkaline reagent to obtain a product;
(3) and (3) reacting the product obtained in the step (2) with ticagrelor in the presence of a catalyst and an alkaline reagent to obtain a crude polypeptide drug conjugate, and cutting, washing, drying and carrying out chromatographic purification to obtain the polypeptide drug conjugate.
5. The production method according to claim 4, wherein the resin used in the solid-phase synthesis method in step (1) is a 2-chlorotrityl chloride resin.
6. The preparation method according to claim 4, wherein the catalyst in the step (2) and the step (3) is O-benzotriazole-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N, N-diisopropylcarbodiimide.
7. The preparation method of claim 6, wherein the mass ratio of the connecting agent, the O-benzotriazole-tetramethylurea hexafluorophosphate, the 1-hydroxybenzotriazole and the N, N-diisopropylcarbodiimide is (1-2) to (1-2) respectively.
8. The method according to claim 4, wherein the basic reagent in the steps (2) and (3) is N, N-diisopropylethylamine.
9. The production method according to claim 6, wherein the mass ratio of the alkali agent to 1-hydroxybenzotriazole is (1.5-3): 1.
10. The method according to claim 4, wherein the cleavage in the step (3) is a cleavage of the polypeptide from the resin using a cleavage solution.
11. The production method according to claim 10, wherein the cutting fluid is a mixed solution composed of 94.5% by mass of trifluoroacetic acid, 2% by mass of water, 2.5% by mass of 1, 2-ethanedithiol, and 1% by mass of triisopropylsilane.
12. The method according to claim 4, wherein the washing in step (3) is carried out using diethyl ether.
13. The use of the polypeptide drug conjugate according to claim 1 or 2 for the preparation of an anti-tumor drug or for the preparation of a drug for the prevention of acute coronary syndrome.
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