Composition and application thereof in preparing medicine with anti-tumor effect
By combining the substance that can cause cystine deprivation with VSVΔ51 oncolytic virus, the problem of the oncolytic effect of VSVΔ51 oncolytic virus in the prior art is solved, and the purpose of significantly improving the anti-tumor effect is achieved.
Patent Information
- Application Number
- CN202411646005.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-03-18
AI Technical Summary
The existing VSVΔ51 oncolytic virus has not had an ideal oncolytic effect in some tumor cells and is difficult to meet the treatment needs of different tumors.
The substance that can cause cystine deprivation is used in combination with VSVΔ51 oncolytic virus to improve the anti-tumor effect through synergistic effects.
The anti-tumor effect of this composition is significantly higher than that of VSVΔ51 oncolytic virus or cystine deprivation substances alone, and can significantly inhibit the survival rate of tumor cells and tumor growth.
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Figure CN119633125A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition and its application in preparing a drug with anti-tumor effect. Background Art
[0002] Oncolytic viruses are a type of replicable virus that selectively infects and kills tumor cells without damaging normal cells. Oncolytic virotherapy is an innovative tumor-targeted treatment strategy that uses natural or genetically engineered viruses to selectively infect tumor cells and replicate in tumor cells, achieving targeted oncolysis and tumor cell killing without damaging normal cells.
[0003] Vesicular stomatitis virus (VSV) belongs to the Rhabdoviridae family and is a negative-strand RNA virus. VSVΔ51 is a recombinant oncolytic virus that lacks the 51st methionine residue of the M protein of the VSV virus. This amino acid deletion can block the impaired interferon response caused by viral infection in normal cells and increase the safety of VSV in normal tissues. VSVΔ51 has a good application effect in the preparation of anti-tumor drugs. However, different tumors have different sensitivities to the VSVΔ51 oncolytic virus. For some tumors, the oncolytic effect of the VSVΔ51 oncolytic virus is not ideal when used alone.
[0004] Therefore, how to further improve the anti-tumor effect of VSVΔ51 oncolytic virus is a technical problem that technicians in this field need to further solve. Summary of the invention
[0005] In order to overcome at least one technical problem existing in the prior art, the present invention provides an application of a composition in preparing a drug having an anti-tumor effect.
[0006] The technical solution of the present invention is as follows:
[0007] The present invention first provides a composition comprising a substance capable of causing cystine deprivation and an oncolytic virus.
[0008] The inventors surprisingly found in their research that the anti-tumor effect of combining substances that can cause cystine deprivation with oncolytic viruses is much higher than that of VSVΔ51 oncolytic virus alone, and also much higher than that of substances that cause cystine deprivation alone. Combining substances that can cause cystine deprivation with oncolytic viruses can produce a synergistic anti-tumor effect.
[0009] Preferably, the oncolytic virus is selected from VSVΔ51 oncolytic virus.
[0010] Further preferably, the VSVΔ51 oncolytic virus is selected from the Indian strain, and its matrix protein (M)
[0011] No.
[0012] The amino acid methionine at position 51 was knocked out.
[0013] Preferably, the substance capable of inducing cystine deprivation includes: cystine-free or low-cystine culture medium, cystine-free or low-cystine food, agents that increase cystine consumption, agents that reduce cystine absorption or transport, and genetic tools that reduce cystine production or increase cystine consumption.
[0014] Preferably, the cystine content in the low-cystine medium or low-cystine food is less than 400 μM.
[0015] Further preferably, the cystine content is below 350 μM, below 300 μM, below 250 μM, below 200 μM, below 150 μM, below 50 μM or below 20 μM.
[0016] Further preferably, the culture medium is DMEM medium.
[0017] Further preferably, the low-cystine food comprises nut-based food.
[0018] Preferably, the agent that increases cystine consumption or the agent that decreases cystine uptake or transport is a cystine transport inhibitor.
[0019] Preferably, the agent that increases cystine consumption or the agent that decreases cystine absorption or transport is an inhibitor of the gene SLC7A11 or SLC3A2.
[0020] Preferably, the agent that increases cystine consumption or the agent that reduces cystine absorption or transport includes but is not limited to Erastin, Erastin2, IKE, Sorafenib, Sulfasalazine, Glutamate or HG106.
[0021] Preferably, the genetic tool for reducing cystine production or increasing cystine consumption is RNA interference, microRNA, gene editing or knockout material.
[0022] Preferably, in the composition, the amount of the substance capable of causing cystine deprivation and the oncolytic virus is
[0023] for
[0024] 300~600nM:0.001MOI.
[0025] Most preferably, in the composition, the dosage ratio of the substance capable of causing cystine deprivation and the oncolytic virus is 500nM:0.001MOI.
[0026] The present invention also provides an application of the above composition in preparing a medicine with anti-tumor effect.
[0027] Preferably, the combination is used in the preparation of a drug with anti-tumor effect.
[0028] Preferably, the tumor is glioma, liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer,
[0029] Front
[0030] Adenocarcinoma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, or stomach cancer.
[0031] Beneficial effects: The present invention provides a new composition; the present invention combines a substance that can cause cystine deprivation with an oncolytic virus for use, and its anti-tumor effect is much higher than the single use of VSVΔ51 oncolytic virus, and is also much higher than the single use of a substance that causes cystine deprivation. Combining a substance that can cause cystine deprivation with an oncolytic virus for use can produce a synergistic anti-tumor effect. Therefore, the combination of the composition for the preparation of a drug with anti-tumor effect has important application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 This is the experimental result showing that CMD and VSVΔ51 oncolytic viruses significantly increased the morphological lesions of human glioma cell lines.
[0033] Figure 2 This is the experimental result showing that combined treatment of CMD / Erastin and VSVΔ51 oncolytic virus significantly reduced the survival rate of human and mouse glioma cell lines.
[0034] Figure 3 This is the experimental result that the combined treatment of CMD and VSVΔ51 oncolytic virus significantly inhibited the growth of transplanted tumors in glioma cell lines. Figure 3 a, CMD combined with VSVΔ51 oncolytic virus treatment significantly inhibited the growth of glioma cell line transplanted tumors; Figure 3 b, Body weight changes of mice treated with CMD and VSVΔ51 oncolytic virus; Figure 3 c, Hematoxylin-eosin (H&E) staining of major tissues and organs of mice treated with CMD and VSVΔ51 oncolytic virus; Figure 3 d, Immunohistochemical staining (IHC) of tumor tissues of mice treated with CMD and VSVΔ51 oncolytic virus.
[0035] Figure 1 to Figure 3 Explanation of the icons in the figure: CMD: cystine deprivation treatment group; VSVΔ51: VSVΔ51 oncolytic virus treatment group; Erastin: SLC7A11 inhibitor; CMD+VSVΔ51: cystine deprivation and VSVΔ51 combined treatment group; Erastin+VSVΔ51: Erastin and VSVΔ51 combined treatment group. DETAILED DESCRIPTION
[0036] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.
[0037] Example 1 CMD and VSVΔ51 viruses significantly increased the morphological lesions of human glioma cell lines.
[0038] Materials: human glioma cells LNZ308, VSVΔ51 oncolytic virus, high-glucose DMEM medium, CMD medium (high-glucose DMEM medium without cystine, methionine and glutamine), inverted phase contrast microscope.
[0039] method:
[0040] a. Cell culture: Human glioma cells LNZ308 were grown in DMEM complete medium containing 10% FBS, 100U / ml penicillin and 0.1mg / l streptomycin; all cell lines were cultured and passaged in a 5% CO2, 37°C constant temperature sealed incubator (relative humidity 95%), and the growth was observed under an inverted microscope. The cells were passaged approximately every 2-3 days, and cells in the logarithmic growth phase were used for formal experiments.
[0041] b. Cell treatment and morphological observation: Cells in logarithmic growth phase were selected and cell suspension was prepared with DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody). 4 The cells were seeded at a density of 100 μg / well in a 24-well culture plate. After the cells adhered to the wall, they were treated with CMD medium alone, infected with VSVΔ51 virus (MOI=0.001), and treated with virus (MOI=0.001) combined with CMD medium. Blank DMEM medium and no virus were used as controls. After 48 hours, the changes in cell morphology were observed under an inverted phase contrast microscope.
[0042] result:
[0043] like Figure 1As shown, the cell morphology was observed under a phase contrast microscope. LNZ308 cells grew in a monolayer and were closely arranged. The cell morphology of the control group and the CMD group was consistent and there was no significant change. After 48 hours of treatment with the VSVΔ51 virus (MOI = 0.001), the morphology of the cells changed significantly; compared with the cells in the control group and the VSVΔ51 alone treatment group, the number of cells in the CMD+VSVΔ51 combined treatment group was significantly reduced, the cell bodies shrank into a sphere, the refractive index was significantly enhanced, and it was a dead lesion. This also shows that the anti-tumor effect of combining substances that can cause cystine deprivation with oncolytic viruses is much higher than that of VSVΔ51 oncolytic viruses alone, and it is also much higher than that of substances that cause cystine deprivation alone. Combining substances that can cause cystine deprivation with oncolytic viruses can produce a synergistic anti-tumor effect.
[0044] Example 2 Combined treatment of CMD / Erastin and VSVΔ51 oncolytic virus significantly reduced the survival rate of human and mouse glioma cell lines.
[0045] Materials: Human glioma cells LN229 and LNZ308, mouse glioma cells GL261, VSVΔ51 oncolytic virus, high-glucose DMEM medium, CMD medium, Erastin, automatic enzyme-linked detection microplate reader Method:
[0046] a. Cell inoculation and drug treatment: cells in logarithmic growth phase were selected and cell suspension was prepared with DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody). 3×10 3 The cells were inoculated at a density of 1.5447 W / well in a 96-well culture plate. After 12 hours, the cells were completely attached to the wall. The cells were treated with CMD medium alone, infected with VSVΔ51 oncolytic virus (MOI=0.001), treated with VSVΔ51 oncolytic virus (MOI=0.001) combined with CMD medium, and treated with Erastin (500 nM) combined with VSVΔ51 oncolytic virus (MOI=0.001). Blank DMEM medium and no virus were used as controls.
[0047] b. MTT reacts with succinate dehydrogenase in cells: After 48 hours of culture, add 20 μl (5 mg / ml) of MTT to each well and continue incubation for 1 to 4 hours. At this time, granular blue-purple formazan crystals formed in living cells can be observed under a microscope.
[0048] c. Dissolve the formazan particles: Carefully remove the supernatant, add DMSO 100 μl / well to dissolve the formed crystals, shake on a micro-oscillator for 5 minutes, and then measure the optical density (OD value) of each well on an enzyme-linked detector at a wavelength of 490 nm. Repeat each group of experiments 3 times. Cell survival rate = OD value of drug-treated group / OD value of control group × 100%.
[0049] d. Graphpad software was used for data analysis, with groups as the horizontal axis and relative cell survival rate as the vertical axis.
[0050] Marking
[0051] Bar graph. The relative cell survival rate was calculated by dividing the survival rate of cells after various treatments by the survival rate of the control group with blank DMEM medium and without virus treatment, and expressed as a multiple. One-way ANOVA statistics indicated that * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 when there is a statistical difference.
[0052] result:
[0053] like Figure 2 As shown in a and 2b, VSVΔ51 oncolytic virus (MOI = 0.001) alone had a small inhibitory effect on the survival of tumor cells LN229, LNZ308 and GL261, and the relative cell survival rate of tumor cells reached more than 70%. The relative cell survival rates of tumor cells in the treatment group were still as high as 95.1% / 95.1%, 91.4% / 97%, and 75.9% / 72.5%, respectively. However, when CMD or Erastin was used in combination with VSVΔ51 oncolytic virus (MOI = 0.001), the relative cell survival rates of tumor cells dropped significantly to 45.9% / 42.6%, 63.5% / 31.8%, and 28.7% / 44.4%, respectively. Compared with single-use treatment, the combined use of CMD oncolytic Erastin and VSVΔ51 virus (MOI = 0.001) significantly reduced the survival rate of tumor cells, indicating that cystine deprivation can enhance the oncolytic effect of VSVΔ51 virus at the cellular level. This also shows that the anti-tumor effect of combining substances that can cause cystine deprivation with oncolytic viruses is much higher than the single use of VSVΔ51 oncolytic virus, and also much higher than the single use of substances that cause cystine deprivation. Combining substances that can cause cystine deprivation with oncolytic viruses can produce a synergistic anti-tumor effect.
[0054] Example 3 Combined treatment of CMD and VSVΔ51 oncolytic virus significantly inhibited the growth of transplanted tumors in glioma cell lines
[0055] Materials: control feed (0.43% w / w methionine, 0.40% w / w cystine), CMD feed (0.15% w / w methionine, 0% w / w cystine), VSVΔ51 oncolytic virus, glioma cell line GL261, and 5-week-old female BALB / c nude mice.
[0056] method:
[0057] a. This experiment adopts a randomized, single-blind design.5 GL261 cells were injected into the skull of 5-week-old BALB / c nude mice (2 mm to the right and 3 mm above the lambdoid suture). When the nude mice showed behavioral changes, they were divided into groups, including the untreated control group, the CMD feed group, and the VSVΔ51 oncolytic virus infection group (VSVΔ51 oncolytic virus 1×10 7 PFU / time) and CMD / VSVΔ51 combined group. The control group feed and CMD feed (mouse food intake 3-5g / day) were replaced one day before virus injection, and then VSVΔ51 oncolytic virus was injected for 6 consecutive days.
[0058] bH&E staining: On the second day after the administration, the mice were killed, the brain tissue was separated, paraffin sections were made, the sections were baked at 60°C for 30 minutes, dewaxed and hydrated; the sections were stained with hematoxylin for 3-5 minutes, rinsed and dehydrated with 95% alcohol for 1 minute, stained with eosin for 15 seconds, and finally dehydrated and sealed. Image J was used to quantify the tumor area and brain tissue area, and the relative percentage of the tumor was plotted.
[0059] cH&E staining: On the second day after the end of drug administration, the mice were killed, the brain tissues were separated, and paraffin sections were made. The sections were baked at 60°C for 30 min, dewaxed and hydrated; they were stained with hematoxylin solution for 3-5 min, rinsed, dehydrated with 95% alcohol for 1 min, stained with eosin solution for 15 s, and finally dehydrated and sealed.
[0060] d. Immunohistochemical analysis (IHC): mice were killed on the second day after the end of administration, brain tissues were separated, paraffin sections were made, and the sections were baked at 60°C for 30 minutes, dewaxed and hydrated; 3% methanol-H2O2 was used for 20 minutes to inactivate endogenous peroxidase; the sections were immersed in boiling 0.01M citrate buffer, treated at low heat for 6 minutes, stopped for 6 minutes, repeated four times, and then naturally cooled to room temperature, antigen repair, and blocked with normal non-immune animal serum for 20 minutes; the blocking solution was discarded, and primary antibodies (Ki67, Cleaved-caspase3) were added to completely cover the specimens, and kept at 4°C overnight; the sections were rewarmed at 37°C for 45 minutes, and the DAB ready-to-use detection system was used for color development, hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing; dehydration, transparency, neutral glue sealing, and drying at 50°C for 1 to 2 hours.
[0061] result:
[0062] exist Figure 3As shown in Figure a, compared with the control group, the weight of mice treated with CMD, VSVΔ51 and the combination group was basically the same, indicating that CMD and VSVΔ51 are safe for mice. Compared with the control group and the single-use group, the tumor volume in the combined treatment group was significantly reduced and the relative percentage of the tumor was reduced, indicating that the combined treatment group inhibited the growth of glioma cell line transplanted tumors (Figures b and c). IHC analysis showed that the application of CMD or VSVΔ51 infection group alone could only cause a slight decrease in Ki67 and a slight increase in Cleaved-caspase3 expression, while the combination of CMD and VSVΔ51 group could cause a significant decrease in Ki67 and a significant increase in Cleaved-caspase3 (Figure d). This also shows that the anti-tumor effect of combining substances that can cause cystine deprivation with oncolytic viruses is much higher than the single use of VSVΔ51 oncolytic virus, and is also much higher than the single use of substances that cause cystine deprivation. Combining substances that can cause cystine deprivation with oncolytic viruses can produce a synergistic anti-tumor effect.
Claims
1. A composition, characterized in that Contains substances that cause cystine deprivation and oncolytic viruses.
2. The composition according to claim 1, characterized in that The oncolytic virus is selected from VSVΔ51 oncolytic virus.
3. The composition according to claim 1, characterized in that The VSVΔ51 oncolytic virus is selected from the Indian strain, and the 51st amino acid methionine of its matrix protein (M) is knocked out.
4. The composition according to claim 1, characterized in that The substances that can cause cystine deprivation include: cystine-free or low-cystine culture medium, cystine-free or low-cystine food, agents that increase cystine consumption, agents that reduce cystine absorption or transport, and genetic tools that reduce cystine production or increase cystine consumption.
5. The composition according to claim 1, characterized in that The cystine content in the low-cystine medium or the low-cystine food is less than 400 μM.
6. The composition according to claim 4, characterized in that The agent that increases cystine consumption or the agent that decreases cystine absorption or transport is a cystine transport inhibitor. Preferably, the agent that increases cystine consumption or the agent that decreases cystine absorption or transport is an inhibitor of the gene SLC7A11 or SLC3A2.
7. The composition according to claim 4, characterized in that The agent that increases cystine consumption or the agent that reduces cystine absorption or transport includes but is not limited to Erastin, Erastin2, IKE, Sorafenib, Sulfasalazine, Glutamate or HG106. Preferably, the genetic tool for reducing cystine production or increasing cystine consumption is RNA interference, microRNA, gene editing or knockout material.
8. The composition according to any one of claims 1 to 7, characterized in that The dosage ratio of the substance that can cause cystine deprivation and the oncolytic virus is 300-600nM:0.001MOI.
9. Use of the composition according to any one of claims 1 to 8 in the preparation of a drug having an anti-tumor effect. Preferably, the combination is used in the preparation of a drug with anti-tumor effect.
10. The use according to claim 9, characterized in that: The tumor is glioma, liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer.