Application of PEG derivative in preparation of antisense oligonucleotide medicine
By modifying PEG derivatives on the antonym nucleotide, R-PEG-Aso is formed, and the problem of insufficient anti-tumor drugs in tumor cells and insufficient anti-degradation capabilities in tumor cells is solved. Internal survival of the cells and more significant inhibitory inhibitory of tumor cell migration.
Patent Information
- Application Number
- CN202411293944.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2025-01-28
AI Technical Summary
The specificity and anti-nuclease degradation ability of existing anti-tumor antisense oligonucleotide drugs in tumor cells is insufficient, resulting in unsatisfactory clinical results.
PEG derivatives modify antonym oligonucleotides to form R-Peg-Aso, where R is a hydroxyl (HO) or folic acid (FA) to improve the tumor cells and anti-degrade ability of the drug.
PEG's ASO is extended in tumor cells. It has higher tumor cells and the effect of inhibiting tumor cell migration, which improves the biocompatibility and clinical safety of the drug.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to the application of a PEG derivative in the preparation of nucleotide drugs. Background Art
[0002] Tumor metastasis is the main cause of death in cancer patients. Gene therapy is a potential therapeutic method to inhibit tumor metastasis. Among them, oligonucleotide therapy uses short nucleotide sequences to change the transcription and translation of the target to achieve the expected pharmacological effect, and has been used in clinical research. Currently reported oligonucleotide drugs mainly include antisense oligonucleotides (ASO), small interfering RNA (siRNA) and aptamers. The activation of oligonucleotides is a complex process that requires oligonucleotides to enter tumor cells; reach effective concentrations at the target site and hybridize with the target. Unmodified oligonucleotides are usually easily degraded by nucleases, resulting in failure. Chemical modification, nanocarriers and polymer coupling are the directions for improving oligonucleotides. Among them, Pegaptanib, which has been approved by the FDA for use in the market, is a polyethylene glycol-coupled oligonucleotide drug. Pegaptanib is an aptamer drug among oligonucleotides, targeting the protein vascular endothelial growth factor overexpressed on the cell surface, blocking the pathological process mediated by vascular endothelial growth factor. Aptamer drugs are limited by limited targets and high-demand screening technologies.
[0003] Antisense oligonucleotides degrade target genes based on the principle of complementary base pairing, and have the advantages of wide applicability and high affinity for targets. The 15 oligonucleotide therapies currently in clinical trials are all antisense oligonucleotide drugs. However, the clinical effect of ASO is not ideal, and there are obvious problems with tumor cell specificity and in vivo circulation time. Therefore, it is still necessary to study new antisense oligonucleotide drugs to improve resistance to nuclease degradation, tumor cell specificity and cell internalization ability.
[0004] Polyethylene glycol (PEG) has good amphiphilicity and biocompatibility, low toxicity, and is one of the few synthetic polymers approved by the US FDA for use in drugs. It has been widely used in the synthesis and modification of compounds to improve the physical and chemical properties and biological activity of compounds. Polyethylene glycol (PEG) modification technology is a new drug delivery technology that has developed rapidly in recent years. Summary of the invention
[0005] In view of the technical problems existing in the existing anti-tumor antisense oligonucleotide drugs, the present invention proposes an application of a PEG derivative in the preparation of an antisense oligonucleotide drug, wherein the PEG derivative is R-PEG-ASO, wherein R is hydroxyl (HO) or folic acid (FA).
[0006] PEGylated ASO has good tumor cell specificity and resistance to nuclease degradation, and can survive in tumor cells for a longer time; it can selectively act on tumor cells, inhibit the migration of tumor cells, and thus treat cancer.
[0007] In some embodiments, R of R-PEG-ASO is folate.
[0008] FA-PEG-ASO has a more significant effect in inhibiting tumor cell migration, has good biocompatibility, and can survive in tumor cells for a longer time.
[0009] In some embodiments, the concentration of the antisense oligonucleotide drug is 0-50 nM.
[0010] In some embodiments, the concentration of the antisense oligonucleotide drug is 5 nM.
[0011] In some embodiments, the antisense oligonucleotide drug is in the form of a solution, suspension, tablet, capsule, granule, injection or infusion.
[0012] The antisense oligonucleotide drug obtained in the present application can effectively inhibit tumor cell migration, has high safety and few side effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 : is a bar graph of cytotoxicity of different embodiments, wherein a is the cytotoxicity of HO-PEG-ASO, and b is the cytotoxicity of FA-PEG-ASO.
[0014] Figure 2 The figure shows the survival status and relative value of fluorescence intensity of different examples in MCF-7 cells.
[0015] Figure 3 The figure shows the survival status of the cells in MCF-7 at different times in different embodiments.
[0016] Figure 4 It is the qRT-PCR analysis diagram of the antisense activity of different examples in MCF-7 cells.
[0017] Figure 5 The figure shows how different embodiments affect the migration ability of MCF-7 cells.
[0018] Figure 6 This is how different concentrations affect the migration ability of MCF-7 cells according to an embodiment of the present invention. DETAILED DESCRIPTION
[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. Some of the terms involved in this application have the following meanings: The term "and / or" is intended to include any and all combinations of one or more of the associated listed items.
[0021] The following is a detailed description in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, other components except inevitable impurities are not included.
[0022] Unless otherwise specified, the reagents and instruments used in the examples are routinely selected in the art.
[0023] The experimental methods without specific conditions in the examples are carried out according to conventional conditions, such as the conditions described in the literature, books or methods recommended by the manufacturer.
[0024] In the present invention, ASO refers to antisense oligonucleotide drugs.
[0025] In the present invention, FA means folic acid.
[0026] In the present invention, PEG means polyethylene glycol.
[0027] In the present invention, miRNA-21 is a type of oncogene that is overexpressed in cancer cells and promotes the migration and proliferation of tumor cells.
[0028] In the present invention, DMSO means dimethyl sulfoxide.
[0029] In the present invention, DMEM medium is a medium containing various amino acids and glucose.
[0030] In the present invention, MCF-7 cells are breast cancer cells.
[0031] Example 1
[0032] Preparation methods of HO-PEG-ASO and FA-PEG-ASO
[0033] The fully complementary sequence of miRNA-21 was designed as the ASO sequence, and the thiol group was modified at its end. The anti-miRNA-21 ASO was coupled with PEG through the addition reaction of the thiol group and the maleimide at the end of PEG to obtain HO-PEG-ASO; the anti-miRNA-21 ASO was coupled with PEG modified with folic acid to obtain FA-PEG-ASO.
[0034] Example 2
[0035] Biocompatibility testing
[0036] The culture conditions of MCF-7 cells are DMEM medium containing 10% fetal bovine serum and 1% double antibody, and a humid environment containing 5% carbon dioxide at 37°C. MCF-7 cells were cultured in a 96-well plate. After the cells adhered to the surface of the wells, they were incubated with different concentrations of HO-PEG-ASO and FA-PEG-ASO for 24 hours, and then MTT was added for another 4 hours. After the culture medium was poured out, 150 μL DMSO was added, and the ultraviolet absorption value at 570 nm was tested using an enzyme marker.
[0037] MTT cytotoxicity assay: The cytotoxicity of HO-PEG-ASO and FA-PEG-ASO was investigated by MTT to evaluate the biocompatibility of HO-PEG-ASO and FA-PEG-ASO. Figure 1 It can be seen that after incubating cells with 1-50 nM HO-PEG-ASO and FA-PEG-ASO for 24 hours, the cell survival rate still reached more than 80%, indicating that HO-PEG-ASO and FA-PEG-ASO have good biocompatibility.
[0038] Example 3
[0039] Antisense activity studies
[0040] The concentration of HO-PEG-ASO and FA-PEG-ASO was selected as 10nM for subsequent cell experiments. The end of ASO was modified with fluorescent dye Cy5, and the cell internalization ability of HO-PEG-ASO and FA-PEG-ASO was analyzed by the fluorescence intensity of Cy5 in cells. Laser confocal microscopy was used to detect the changes in the fluorescence intensity of HO-PEG-ASO and FA-PEG-ASO in MCF-7 cells over time.
[0040] The distribution of the ASO within the cell and the effective time required to search for and hybridize with the target RNA sequence are another requirement for the use of ASOs. Figure 3The results showed that at 5 h, a large amount of FA-PEG-ASO was still observed in MCF-7 cells. Cell imaging results showed that the conjugation of ASO with PEG modified with folic acid could significantly improve the uptake capacity and intracellular survival time of PEG-ASO in tumor cells.
[0041] from Figure 4 The results of qRT-PCR showed that the content of target miRNA-21 in MCF-7 cells treated with two PEG-ASOs was significantly decreased. Moreover, FA-PEG-ASO showed stronger miRNA-21 cleavage than HO-PEG-ASO, which was attributed to the higher cellular uptake rate of FA-PEG-ASO ( Figure 2 ) and longer cell survival time ( Figure 3 ).
[0042] Example 4
[0043] Scratch test
[0044] The scratch test was performed in a six-well plate. An equal number of cells were seeded into each well. When the cells had basically filled the entire well, a notch was made on the plate with a pipette tip. Then, different concentrations of HO-PEG-ASO and FA-PEG-ASO were incubated, and the migration results of the cells at specific time points were observed and recorded under a microscope.
[0045] The scratch assay was used to evaluate the effect of PEG-ASO on cell migration ability. Figure 5 The results showed that both PEG-ASO conjugates inhibited the migration of MCF-7 cells. Moreover, FA-PEG-ASO had a higher ability to inhibit the migration of MCF-7 cells than HO-PEG-ASO. The effect of 0-10 nM FA-PEG-ASO on the migration of MCF-7 cells was further determined to further verify its antisense activity. Figure 6 It can be seen that the migration ability of MCF-7 cells decreases with the increase of FA-PEG-ASO concentration. When the concentration of FA-PEG-ASO reaches 5nM, the migration ability of MCF-7 cells is basically inhibited.
[0046] In summary, both HO-PEG-ASO and FA-PEG-ASO showed strong target RNA cleavage ability and superior ability to inhibit tumor cell migration. Among them, the cell internalization ability of folic acid-modified PEG-ASO was significantly higher than that of HO-PEG-ASO. Cell imaging results showed that the cell internalization rate of folic acid-modified PEG-ASO was 5 times higher than that of PEG-ASO without folic acid modification. The clearance rate of folic acid-modified PEG-ASO on target RNA was also higher than that of PEG-ASO without folic acid modification, and the inhibitory effect on cell migration was also stronger than that of HO-PEG-ASO. This shows that both HO-PEG-ASO and FA-PEG-ASO can treat tumor cells, and FA-PEG-ASO has a better effect.
Claims
1. Use of a PEG derivative in the preparation of an antisense oligonucleotide drug, characterized in that: The PEG derivative is R-PEG-ASO, wherein R is hydroxyl or folic acid.
2. The use according to claim 1, characterized in that in, R stands for folic acid.
3. The use according to claim 1, characterized in that: The concentration of the antisense oligonucleotide drug is 0-50 nM.
4. The use according to claim 3, characterized in that: The concentration of the antisense oligonucleotide drug was 5 nM.
5. The use according to claim 1, characterized in that: The antisense oligonucleotide drug is in the form of a solution, a suspension, a tablet, a capsule, a granule, an injection or an infusion.