CN111529714B

CN111529714B - Full-phosphorothioate modified aptamer drug conjugate and preparation method and application thereof

Info

Publication number: CN111529714B
Application number: CN202010537824.8A
Authority: CN
Inventors: 谭蔚泓; 王雪强; 杨秋霞; 符婷
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2022-11-15
Anticipated expiration: 2040-06-12
Also published as: CN111529714A

Abstract

The invention relates to the field of medicines, in particular to a wholly thiophosphate-modified aptamer medicine conjugate and a preparation method and application thereof. The invention provides a total phosphorothioate modified aptamer drug conjugate, which comprises a drug molecule group and a phosphorus-sulfur bond substituted aptamer segment, wherein the drug molecule group is selected from a mitomycin C group, and the drug molecule group is connected with the phosphorus-sulfur bond substituted aptamer segment through a connecting group. The aptamer drug conjugate modified by the total phosphorothioate has all the advantages of a aptamer, the enzyme digestion resistance of the aptamer-mitomycin C conjugate is greatly improved through the phosphorothioate modification of the phosphate skeleton of the aptamer fragment, the blood circulation half-life of the aptamer-mitomycin C conjugate is prolonged, the stability is improved, the original targeting property and specificity of the aptamer-mitomycin C conjugate are retained, and the aptamer-mitomycin C conjugate has good industrialization prospects.

Description

Full-phosphorothioate modified aptamer drug conjugate and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a wholly thiophosphate-modified aptamer medicine conjugate and a preparation method and application thereof.

Background

Mitomycin (Mitomycin) is an antitumor antibiotic separated from a culture solution of streptomyces capitata, is effective on various solid tumors, can form cross-linking with a double-helix structure of DNA, destroys the structure and the function of the DNA, inhibits the replication of the DNA in a proliferation stage, has a killing effect on cells in the proliferation stage, and can also act on cells in a stationary stage, thereby inhibiting tumor cells, and is clinically suitable for digestive tract cancers such as gastric cancer, intestinal cancer, liver cancer, pancreatic cancer and the like.

Aptamer (Aptamer) is an oligonucleotide fragment obtained from a nucleic acid molecule library by using in vitro screening technology, i.e., exponential enrichment ligand phylogenetic technology (SELEX), and can specifically bind to a target molecule. Compared with the commonly used targeting molecule antibody, the aptamer has the advantages of short screening period, easy synthesis and modification, low preparation cost, stronger stability, wide target range, low toxicity and immunogenicity, strong tissue penetrability and wider application prospect in tumor diagnosis and treatment. Besides being used as an anti-tumor medicament, the aptamer can be further used as a targeting molecule to be connected with small-molecule anti-tumor medicaments which have excellent anti-tumor performance but do not have targeting property, the targeting capability is endowed, target proteins with high expression on the surface of tumor cells can be specifically identified, and the medicaments are conveyed to tumor parts, so that the precise release of the medicaments is realized, the anti-tumor effect is improved, and the toxic and side effects on normal tissues are reduced.

However, in practical applications, aptamers present a series of troublesome problems. The most important of which is its instability in vivo. Naked natural nucleic acid is easily recognized and digested by various nucleases in vivo in blood circulation, so that the naked natural nucleic acid is possibly degraded before reaching a targeted lesion part, and the efficacy of the naked natural nucleic acid is greatly reduced. To solve this problem, researchers have resorted to various methods of modifying nucleic acids to protect them from enzymatic cleavage. The most common of these are chemical modifications of nucleic acids, such as chemical modifications of the nucleic acid backbone, chemical modifications of the sugar rings and chemical modifications of the bases.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a phosphorothioate-modified aptamer drug conjugate, and a preparation method and use thereof, which are used to solve the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a phosphorothioate-modified aptamer drug conjugate, comprising a drug molecule group and a phospho-sulfur bond-substituted aptamer segment, wherein the drug molecule group is selected from mitomycin C group, and the drug molecule group is connected to the phospho-sulfur bond-substituted aptamer segment through a linking group.

In some embodiments of the invention, the structural formula of the drug molecule group is as follows:

in some embodiments of the invention, the phosphosulphur bond-substituted aptamer segment is in particular a segment in which the phosphooxygen bond is substituted by a phosphosulphur bond.

In some embodiments of the invention, the polynucleotide sequence of the aptamer fragment comprises the sequence shown as SEQ ID No. 1.

In some embodiments of the invention, the 5' end of the phosphosulphur-bond-substituted aptamer fragment is linked to the linker group via an-S-S-bond.

In some embodiments of the invention, the chemical structure of the linking group is as follows:

in some embodiments of the invention, the structural formula of the phosphorus sulfur bond substituted aptamer drug conjugate is as follows:

in another aspect, the present invention provides a method for preparing the above-mentioned full phosphorothioate modified aptamer drug conjugate, comprising: and (3) connecting a drug molecule with the nucleic acid fragment substituted by the phosphorus-sulfur bond so as to provide the complete thiophosphate modified aptamer drug conjugate.

In some embodiments of the present invention, the preparation method specifically comprises: modifying a drug molecule into a connecting molecule, and connecting the drug molecule modified with the connecting molecule with a nucleic acid fragment substituted by a phosphorus-sulfur bond to provide the phosphorothioate-modified aptamer drug conjugate.

In another aspect, the present invention provides the use of the above-mentioned fully phosphorothioate-modified aptamer drug conjugate in the preparation of a medicament.

Drawings

FIG. 1 is a schematic diagram showing the identification results of mitomycin C aptamer conjugates in example 1 of the present invention.

FIG. 2 is a schematic diagram showing the experimental results of tumor cell targeted uptake capacity in example 2 of the present invention.

FIG. 3 is a graph showing the results of in vitro targeted toxicity experiments in example 3 of the present invention.

FIG. 4 is a schematic diagram showing the results of pharmacokinetic measurements of rat in example 4 of the present invention.

FIG. 5 is a graph showing the results of in vivo imaging experiments on mice in example 5 of the present invention.

FIG. 6 is a schematic diagram showing the chemical structure of the phosphorus-sulfur bond-substituted aptamer fragment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

The inventors of the present invention have surprisingly found, after a large number of practical studies, that the in vivo half-life of a nucleic acid aptamer drug conjugate can be effectively improved by modifying the phosphate backbone of the nucleic acid aptamer with phosphorothioate, so that the nucleic acid aptamer drug conjugate has better drug stability, and thus the present invention has been completed.

The invention provides a full phosphorothioate modified aptamer drug conjugate, which comprises a drug molecule group and a phosphorus-sulfur bond substituted aptamer segment, wherein the drug molecule group is selected from a mitomycin C group, and the drug molecule group is connected with the phosphorus-sulfur bond substituted aptamer segment through a connecting group. The aptamer segment substituted by phosphorus-sulfur bonds specifically refers to that phosphorus-oxygen bonds in the molecular structure of the aptamer segment are at least partially or completely replaced by phosphorus-sulfur bonds, the specific structural change is shown in fig. 6, in the figure, the left figure is the structure with the phosphorus-oxygen bonds being not replaced, and the right figure is the structure with the phosphorus-oxygen bonds being replaced by the phosphorus-sulfur bonds. The aptamer fragment substituted by the phospho-sulfide bond can be coupled with mitomycin C, and the modification method can greatly improve the enzyme digestion resistance of the aptamer-mitomycin C conjugate, prolong the blood circulation half-life period of the conjugate, improve the endocytosis capacity of the conjugate on target cells, and further improve the drug effect of the conjugate in tumor treatment.

The aptamer drug conjugate provided by the invention can comprise a drug molecule group, and the drug molecule group can be a mitomycin C group formed by a mitomycin C molecule. In a preferred embodiment of the present invention, the drug molecule may specifically be a compound having the following chemical formula:

the drug molecule may comprise a cyclic ethylamine such that it can react with the linker molecule via the cyclic ethylamine group to form a drug molecule group. In a preferred embodiment of the present invention, the chemical structural formula of the drug molecule group is as follows:

the aptamer drug conjugate provided by the invention can comprise a aptamer segment substituted by a phosphorus-sulfur bond. The selection of a particular sequence of the aptamer fragment, which may include the sequence shown in SEQ ID No. 1: XQ-2d,5 '-ACTCATAGGGGTTAGGGGCTGCTGGCCAGATACTCAGAGTGGTAGGGTTACTATGAGC-3' (SEQ ID NO. 1). The aptamer fragment is typically targeted to its corresponding protein, e.g., the aptamer fragment shown above can target CD71 protein, which is typically highly expressed on the surface of a variety of tumor cells, to achieve specific targeting of tumor cells. As described above, the phosphorus-sulfur bond-substituted aptamer segment specifically means that the phosphorus-oxygen bonds in the molecular structure of the aptamer segment are all substituted by phosphorus-sulfur bonds. The phospho-thio bond-substituted aptamer fragment can be generally formed by chemical modification of a suitable nucleic acid fragment, and suitable modification methods are known to those skilled in the art, and for example, phosphorothioate modification can be performed on the phosphate backbone of a nucleic acid fragment, and a specific preparation method can be referred to the preparation method of M.E.Dugras, B.J.Merlow.phosphorothioate oligonucleotide: CN. The aptamer fragments can also be generally formed from nucleic acid fragments that are modified at the 5 'end with an-SH, such that an-S-S-linkage can be formed between the 5' end of the aptamer fragment and the linking group. Methods for modifying the 5 'end of a nucleic acid fragment with-SH are known to those skilled in the art, and for example, a nucleic acid fragment may be modified with a C6 thiol phosphoramidite monomer to obtain a nucleic acid fragment modified at the 5' end with-SH. In a preferred embodiment of the present invention, the C6 thiol phosphoramidite monomer can be modified at the 5' end by using a DNA solid phase synthesis method, and the chemical structure of the C6 thiol phosphoramidite monomer is shown as follows:

the hydroxyl on the sugar ring at the 5' end of the nucleic acid fragment is crosslinked with a phosphoramidite monomer, and the reaction equation is as follows:

wherein the-CH 2-linked curve portion on the five-membered heterocyclic branch represents the remainder of the nucleic acid fragment and the filled circle represents the solid support CPG. The DNA phosphate skeleton is modified by sulfydryl, the prepared product is deprived of a protecting group, and a nucleic acid fragment with the 5 'end modified by-SH is obtained, and is further connected with a connecting group through the-SH group, so that the 5' end of the aptamer fragment is connected with the connecting group through an-S-S-bond, and the structural formula of the aptamer fragment connected by the-S-S-bond is as follows:

the aptamer drug conjugate provided by the invention can comprise a connecting group, and the structural formula of the connecting group is as follows:

the connecting group is mainly used for preventing the targeting property of the aptamer from being influenced. The two ends of the connecting group can be respectively connected with a drug molecule group and a nucleic acid aptamer segment substituted by a phosphorus-sulfur bond. Specifically, the 5' end of the aptamer fragment substituted by the phosphorus-sulfur bond is connected with a connecting group through-S-S-bond, and the drug molecule group can be connected with the connecting group through-COO-bond. The linking group may generally be a group formed by a suitable linking molecule with the drug molecule and the nucleic acid fragment, for example, the linking molecule may comprise

Thereby allowing attachment of drug molecules and nucleic acid fragments to form attachment groups. In a preferred embodiment of the present invention, the linker molecule may specifically be a compound having the following chemical formula:

in a more preferred embodiment of the present invention, the chemical structural formula of the phosphorothioate-modified aptamer drug conjugate is as follows:

wherein the portion of the curve to which-S-is attached is a fragment of the aptamer substituted by a phospho-thio bond.

In a second aspect, the present invention provides a method for preparing a phosphorothioate-modified aptamer drug conjugate, comprising: the method for providing the phosphorothioate-modified aptamer drug conjugate can be specifically referred to the literature methods given above.

In the preparation method provided by the present invention, the preparation method may specifically include: 1) Modifying the drug molecule with a linker molecule; 2) And reacting the drug molecule modified with the connecting molecule with the nucleic acid fragment substituted by the phosphorus-sulfur bond to provide the full phosphorothioate modified aptamer drug conjugate.

In the preparation method provided by the invention, the drug molecule is usually mitomycin C molecule, and the mitomycin C molecule usually contains a dimethylimine group (aziridine). Methods for modifying a pharmaceutical molecule containing a diimine group (aziridine) with a suitable linker molecule to provide a drug molecule modified with a linker molecule will be known to those skilled in the art and may be, for example: the drug molecule and the linker molecule are reacted in the presence of a catalyst, which may be an organic base in general, and more specifically, 4-lutidine or the like. For another example, the reaction may be carried out in the presence of a suitable reaction solvent, and specifically, DMF and the like may be used. For another example, the reaction may be carried out at room temperature or under heating, and the specific reaction temperature may be room temperature.

In the preparation method provided by the present invention, the method for reacting the drug molecule modified with the linker molecule with the nucleic acid fragment substituted with the phosphorus-sulfur bond to provide the phosphorothioate-modified aptamer drug conjugate is known to those skilled in the art, for example, the drug molecule modified with the linker molecule and the nucleic acid fragment substituted with the phosphorus-sulfur bond may be mixed well. For another example, the reaction may be carried out in the presence of a suitable reaction solvent, specifically DMF and the like. For another example, the reaction may be carried out at room temperature or under heating, and the specific reaction temperature may be room temperature.

In a preferred embodiment of the present invention, the reaction equation for modifying a drug molecule with a linker molecule to provide a drug molecule modified with a linker molecule is as follows:

in a preferred embodiment of the present invention, in the preparation method, the reaction equation of reacting a drug molecule with a nucleic acid fragment substituted by a phosphorus-sulfur bond to provide the phosphorothioate-modified aptamer drug conjugate is as follows:

in a third aspect, the invention provides the use of the phosphorothioate-modified aptamer drug conjugate provided in the first aspect of the invention in the preparation of a medicament. The total phosphorothioate modified aptamer drug conjugate provided by the invention has good specificity and targeting property for target cells (for example, tumor cells, specifically human pancreatic cancer cells and the like, and more specifically PL45 cells and the like), and the aptamer drug has good target uptake capacity and target killing capacity for the tumor cells and has longer retention time at tumor sites, so that the aptamer drug conjugate can be used as a tumor treatment drug.

The aptamer-mitomycin C conjugate has the advantages of all the aptamers, the enzyme digestion resistance of the aptamer-mitomycin C conjugate is greatly improved through phosphorothioate modification of the phosphate skeleton of the aptamer fragment, the blood circulation half-life of the aptamer-mitomycin C conjugate is prolonged, the stability is improved, the original targeting property and specificity of the aptamer-mitomycin C conjugate are reserved, and the aptamer-mitomycin C conjugate has a good industrialization prospect.

The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Example 1

Preparation of 4-nitrophenyl-4- (2-dimercaptopyridinyl) ethyl carbonate:

a50 mL three-necked round-bottomed flask was charged with 88mg (0.4 mmol) of 2,2' -dimercaptopyridine, 31.3mg (0.4 mmol) of 2-mercaptoethanol, and 10mL of dichloromethane. Stir at rt for 2h and monitor the progress of the reaction by TLC. Removing dichloromethane by rotary evaporation under reduced pressure, and purifying by silica gel chromatography to obtain 20mg 2- (2-dimercaptopyridyl) ethanol as yellowish white oily liquid with a yield of 53%.

To a 50mL three-necked round bottom flask was added 18.7mg (0.1 mmol) of 2- (2-dimercaptopyridinyl) ethanol, 10mL of anhydrous dichloromethane, 40mg (0.2 mmol) of p-nitrophenyl chloroformate, 25.9mg (0.2 mmol) of N, N' -diisopropylethylamine, and 1.22mg (catalytic amount) of 4-dimethylaminopyridine, and the reaction was stirred overnight at room temperature and monitored by TLC. After the reaction was completed, 0.01M hydrochloric acid solution was added to neutralize excess alkali, followed by extraction with dichloromethane/saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, and then dichloromethane was evaporated under reduced pressure, and silica gel column chromatography was performed to obtain 19.35mg of 4-nitrophenyl-4- (2-dimercaptopyridine) ethyl carbonate as a yellowish white oily liquid in 55% yield.

b. Preparation of mitomycin C-4- (2-pyridyldithio) ethyl carbonate amide:

a50 mL three-necked round-bottomed flask was charged with 19.35mg (0.055 mmol) of 4-nitrophenyl-4- (2-dimercaptopyridine) ethyl carbonate, 16.7mg (0.05 mmol) of mitomycin, and a catalytic amount of 4-dimethylaminopyridine, and 10mL of N, N-dimethylformamide was added thereto under nitrogen protection, and the mixture was stirred at room temperature overnight, and the progress of the reaction was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane/saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove dichloromethane, and purified by silica gel chromatography to give 24.7mg of mitomycin C-4- (2-dimercaptopyridine) ethylcarboxamide as a black solid at a yield of 90%.

c. Preparation of mitomycin C aptamer conjugate:

mitomycin C-4- (2-dimercaptopyridine) ethylcarboxamide 1mg was dissolved in 200. Mu.L of N, N-dimethylformamide, and 200. Mu.L of 30. Mu.M thiol-modified thioacid aptamer solution and thiol-modified non-thioacid aptamer solution (SEQ ID NO. 1) (purchased from Shanghai Biotechnology, ltd.) were added and shaken at 37 ℃ for 24 hours. After the reaction, the product is purified by a reverse preparative column, the obtained target product is subjected to freeze drying to obtain a product, the product is dissolved in ultrapure water to obtain 144 μ L of 25 μ M, the yield is 60%, and the mass spectrometry result is shown in fig. 1, wherein fig. 1A is a schematic diagram of the relative molecular mass of an unsulfothioated aptamer drug conjugate (ApDC), and fig. 1B is a schematic diagram of the relative molecular mass of a thioated aptamer drug conjugate (S-ApDC).

Example 2

The target uptake capacity of the thioacid aptamer-mitomycin C conjugate by tumor cells:

the ability of the thioate aptamer drug conjugate S-ApDC (prepared in example 1) to selectively enter tumor cells was further examined, while the control sequence drug conjugate CpDC (5. PL45 cells or MCF-7 cells are digested and inoculated into a 12-well plate, the density is 10 ten thousand per dish, after being cultured in an incubator at 37 ℃ for 24 hours, the cells are washed three times by DPBS, then 200 mu L of single strands (XQ-2 d or S-XQ-2d, respectively, non-thioated aptamer and thioated aptamer, neither coupled with drug, SEQ ID NO. 1) and S-ApDC/CpDC containing 250nM Cy5 marker are respectively added, after being incubated at 37 ℃ for 2 hours, the cells are washed 3 times by DPBS and then digested for 1-2min by pancreatin, and the detection is carried out by flow (FACS BD Verse), and the results are shown in FIG. 2, the left graph is a graph of the experimental results of PL45 cells, and the right graph is a graph of the experimental results of MCF-7 cells. For PL45 cells, both S-ApDC and S-XQ-2d showed greater endocytosis than XQ2d, while CpDC was the least endocytosis. All endocytic capacity was relatively poor for MCF-7 cells.

Example 3

In vitro targeted toxicity experiments:

cleaning cells with DPBS, adding 200 mu L of pancreatin for digestion for 2-3min, blowing and resuspending the cells into a culture medium, counting the number of the cells by a counting plate, inoculating the cells into a 96-well plate, wherein each hole comprises 5000 cells, culturing for 24h, adding fresh culture media containing mitomycin C, S-ApDC and ApDC in a series of concentration gradients respectively, and culturing for 72h. Then, the CCK-8 kit is used for detecting the cell viability, and the result is shown in figure 3, wherein the left figure is a schematic diagram of the PL45 cell experiment result, and the right figure is a schematic diagram of the MCF-7 cell experiment result. For the target cell PL45, the toxicity of ApDC is stronger than that of mitomycin C alone, and the toxicity of S-ApDC is equivalent to that of mitomycin C. Meanwhile, for non-target cells MCF-7, apDC toxicity is similar to that of a single drug, no enhancement effect is achieved, and S-ApDC hardly shows toxicity, so that the safety of S-ApDC is further proved.

Example 4

Rat pharmacokinetic testing:

to verify whether the thio-modification can increase the blood circulation half-life of the conjugate, further rat pharmacokinetic experiments were performed. About 130g of rats are injected with 200 muL of Cy 5-labeled S-ApDC and ApDC 100 muM into tail veins respectively, then about 1mL of blood is taken from eyeballs at 0,0.5,1,2,4,8 and 12h, the fluorescence intensity of the blood is detected by a small animal imager, the fluorescence intensity of the sample at different time points is calculated by comparing with a standard curve of the fluorescence intensity of the blood and the concentration of the sample, and the blood circulation half life is calculated by fitting, as shown in figure 4. The results show that the half-life of the conjugate in rats can be greatly improved after the thiomodification.

Example 5

Mouse in vivo imaging experiments:

in order to verify the in vivo distribution and tumor targeting ability of the conjugate after thiomodification, a mouse in vivo imaging experiment was further performed. Firstly, the mice are implanted with tumor, and about 8 weeks of BALB/c nude mice are respectively injected with 2 x 10^4 PL45 cells subcutaneously, and the imaging is carried out after two weeks of tumor formation. Mu.l of 50. Mu.M Cy 5-labeled S-ApDC and ApDC were injected into the tail vein, respectively, and then small animal imaging was performed at 0,0.5,1,2,4,8,12,24, 48h. As a result, S-ApDC reached the tumor site better and the retention time in vivo was longer, as shown in FIG. 5.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A phosphorothioate-modified aptamer drug conjugate comprises a drug molecule group and a phosphorus-sulfur bond substituted aptamer segment, wherein the drug molecule group is selected from a mitomycin C group, the drug molecule group is connected with the phosphorus-sulfur bond substituted aptamer segment through a connecting group, and the phosphorus-sulfur bond substituted aptamer segment is formed by replacing a phosphorus-oxygen bond in the aptamer segment with a phosphorus-sulfur bond; the polynucleotide sequence of the aptamer fragment comprises a sequence shown as SEQ ID NO. 1; the chemical structural formula of the connecting group is as follows:

2. the aptamer drug conjugate of claim 1, wherein the structural formula of the drug molecule group is as follows:

3. the aptamer drug conjugate of claim 1, wherein the 5' terminus of the phospho-sulfur bond substituted aptamer fragment is linked to the linking group via an-S-bond.

4. The aptamer drug conjugate of claim 1, wherein said phospho-sulfide bond substituted aptamer drug conjugate has the formula:

5. the method for preparing a phosphorothioate-modified aptamer drug conjugate according to any one of claims 1 to 4, comprising: and (3) connecting a drug molecule with the nucleic acid fragment substituted by the phosphorus-sulfur bond so as to provide the complete thiophosphate modified aptamer drug conjugate.

6. The preparation method according to claim 5, wherein the preparation method specifically comprises: modifying a drug molecule into a connecting molecule, and connecting the drug molecule modified with the connecting molecule with a nucleic acid fragment substituted by a phosphorus-sulfur bond to provide the phosphorothioate-modified aptamer drug conjugate.

7. Use of the phosphorothioate-modified aptamer drug conjugate according to any one of claims 1 to 4 for the preparation of a medicament for pancreatic cancer.