Use of pcsk9 inhibitors and oncolytic viruses in the preparation of antitumor drugs

Abstract

The present application relates to the application of PCSK9 inhibitor and oncolytic virus in the preparation of antitumor drugs, and belongs to the technical field of tumor treatment. The present application finds that PCSK9 inhibitor can increase the antitumor effect of oncolytic virus, so as to improve the therapeutic effectiveness of oncolytic virus as an antitumor drug. The present application proves through zoological experiments that the combined application of oncolytic virus and PCSK9 inhibitor can cause the improvement of tumor immune microenvironment, thereby significantly enhancing the inhibition effect on tumor cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor treatment, and particularly relates to application of a PCSK9 inhibitor and an oncolytic virus in preparation of an antitumor drug. BACKGROUND

[0002] Oncolytic virus (OV) is a kind of replicable virus that selectively infects and kills tumor cells without damaging normal cells. Oncolytic virotherapy is an innovative tumor-targeting treatment strategy that uses naturally occurring or genetically engineered viruses to selectively infect tumor cells and replicate in them, achieving the effect of targeted lysis and killing of tumor cells, but without damaging normal cells.

[0003] Oncolytic viruses can be divided into two categories: natural viruses and transgenic viruses. Naturally occurring OV includes reovirus, Newcastle disease virus (NDV), enterovirus and measles virus (MV), etc., while most OV are genetically modified to increase the tropism of the virus to tumor cells, improve the selective replication and lysis potential of the virus, and enhance the host's anti-tumor immunity.

[0004] In the 21st century, after many clinical trials obtained positive results, the OV field has gained considerable attention. So far, four OV drugs have been approved globally.

[0005] Although oncolytic virus therapy has shown potential in various tumor models, not all tumors are sensitive to oncolytic viruses. Some tumors may not be sensitive or resistant to oncolytic virus killing due to their biological characteristics or microenvironment factors. The following are the reasons why some tumors may not be sensitive or resistant to oncolytic viruses:

[0006] 1. Tumor microenvironment resistance: The microenvironment of some tumors may not be conducive to the replication and spread of oncolytic viruses, such as by producing anti-viral proteins or immunosuppressive molecules to limit viral activity;

[0007] 2. Antiviral ability of tumor cells: Some tumor cells may have strong antiviral signaling pathways that can effectively inhibit viral replication, reducing the killing effect of oncolytic viruses;

[0008] 3. Heterogeneity of tumor cells: There may be high heterogeneity within the tumor, with some cell subpopulations being insensitive to oncolytic viruses, limiting treatment effectiveness;

[0009] 4. Immune escape of tumor cells: Some tumor cells may evade the immune system's attack by modulating immune checkpoint molecules such as PD-L1, which can affect the ability of oncolytic viruses to activate anti-tumor immune responses;

[0010] 5. Genetic background of tumor: specific genetic variations or mutations can affect the sensitivity of tumor to oncolytic viruses, for example, certain tumors may have resistance to viral killing due to specific genetic mutations;

[0011] 6. Differentiation state of tumor: the degree of differentiation of tumor cells can affect their sensitivity to oncolytic viruses, undifferentiated or poorly differentiated tumor cells may have higher resistance to viral infection and killing;

[0012] 7. Angiogenesis of tumor: angiogenesis of tumor can affect the delivery efficiency of oncolytic viruses, poor angiogenesis can limit the virus to reach tumor cells, thereby affecting the treatment effect.

[0013] Therefore, it is a technical problem to be solved in the art to improve the tumor cell killing ability of oncolytic viruses. SUMMARY

[0014] The present application aims to overcome the shortcomings of the prior art and provide the use of PCSK9 inhibitors in the preparation of oncolytic virus antitumor synergists or drug resistance reversing agents.

[0015] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0016] In a first aspect, the present application provides the use of PCSK9 inhibitors in the preparation of tumor drugs

[0017] The present application finds that PCSK9 inhibitors have certain antitumor effect, so they can be used in the field of tumor treatment, and their new use is developed.

[0018] As a preferred embodiment, the tumor drug is an antitumor synergist or an antitumor drug resistance reversing agent.

[0019] The present application finds that the combination of PCSK9 inhibitors and oncolytic viruses for treating tumors can significantly improve the therapeutic effect of tumors, indicating that the two have a synergistic effect, therefore, PCSK9 inhibitors can be used as oncolytic virus antitumor synergists to improve the antitumor therapeutic effect of oncolytic viruses.

[0020] In a second aspect, the present application provides a pharmaceutical composition, which comprises: an oncolytic virus and a PCSK9 inhibitor.

[0021] As a preferred embodiment, the oncolytic virus further comprises a recombinant oncolytic virus comprising a nucleotide sequence encoding an anti-PCSK9 antibody. The recombinant oncolytic virus of the present application can integrate the nucleotide sequence encoding the anti-PCSK9 antibody on the oncolytic virus by genetic recombination.

[0022] As a preferred embodiment, the oncolytic virus is selected from at least one or several of the following: an alphavirus, an adenovirus, a vaccinia virus, a measles virus, a vesicular stomatitis virus, and a herpes simplex virus.

[0023] As a preferred embodiment, the alphavirus is selected from at least one of the following: M1 virus and Getah virus.

[0024] The alphavirus used in the embodiments of the present application is M1 virus with the accession number CCTCC V201423 (deposited in China Center for Type Culture Collection on July 17, 2014), and the sequence of M1 is recorded in Genbank Accession No. EF011023, while Getah virus is a virus with up to 97.8% homology with M1 virus (Wen et al. Virus Genes. 2007; 35(3):597-603), both of which have high identity, and at the same time, M1 virus is also classified as a Getah-like virus, so it can be expected that both have the same efficacy.

[0025] As a preferred embodiment, the vesicular stomatitis virus is VSVΔ51 strain.

[0026] The vesicular stomatitis virus (VSV) used in the embodiments of the present application is a non-pathogenic, enveloped, negative-strand RNA rhabdovirus that can infect almost all types of cells, but due to the interferon (IFN) -mediated antiviral response, it cannot cause productive infection in healthy cells. However, IFN signaling defects often occur simultaneously with tumor production. Therefore, VSV can infect and selectively destroy tumor cells with little damage to normal cells, and these characteristics make it an ideal oncolytic virus therapeutic agent. VSV has many advantages as an oncolytic virus, including a short replication cycle, the ability to target multiple cancer cells, the ability to replicate normally under hypoxic conditions in the tumor microenvironment to exert tumor-killing effects, and a small viral genome that is easy to direct modification, but wild-type VSV still has many defects, mainly manifested as potential neurotoxicity. In order to reduce neurotoxicity, VSV strain (i.e. VSVΔ51) can be designed by deleting the methionine at the 51st residue of the matrix protein, and compared with the parent strain, VSVΔ51 has a stronger ability to induce IFN response in cells.

[0027] As a preferred embodiment, the PCSK9 inhibitor is selected from one or several of the following: a substance that specifically binds to PCSK9 molecules, a substance that inhibits the activity of PCSK9 protein, or a substance that degrades PCSK9 protein, or a genetic tool that reduces the level of PCSK9 protein.

[0028] As a preferred embodiment, the substance that specifically binds to the PCSK9 molecule is selected from one or more of Alirocumab, Evolocumab, Tafolecimab, or their derivatives, solvates, tautomers, isomers.

[0029] PCSK9 inhibitors are a class of drugs used to reduce low-density lipoprotein cholesterol (LDL-C) levels in the blood. They work by inhibiting the PCSK9 protein to reduce the degradation of low-density lipoprotein (LDL) receptors, thereby increasing the clearance of LDL-C. Currently, several PCSK9 inhibitors have been approved for clinical treatment on the market:

[0030] Evolocumab: is a fully human IgG2 monoclonal antibody that can bind to PCSK9 protein and block PCSK9-mediated LDL receptor degradation. Clinical studies have shown that Evolocumab can further significantly reduce LDL-C levels by an average of 59% on the basis of statins, and reduce the risk of cardiovascular events by 15%.

[0031] Alirocumab: as a PCSK9 monoclonal antibody, it prevents PCSK9 from interacting with LDL receptors by binding to PCSK9, thereby increasing the clearance of LDL-C. In the FOURIER and ODYSSEY clinical trials, Evolocumab and Alirocumab both showed effective reduction in LDL-C levels and reduction in cardiovascular event risk.

[0032] Tafolecimab is another PCSK9 monoclonal antibody that also works by inhibiting the PCSK9 protein. The ORION study confirmed the effectiveness and safety of Tafolecimab.

[0033] The present application has been proven by experiments that the combination of PCSK9 inhibitors can increase the antitumor spectrum and intensity of oncolytic viruses, therefore, PCSK9 inhibitors can improve the antitumor effect of oncolytic viruses.

[0034] As a preferred embodiment, the gene tool for reducing the level of PCSK9 protein is RNA interference, microRNA, gene editing tool.

[0035] As a preferred embodiment, the RNA interference is Inclisiran or its derivatives, solvates, tautomers, isomers.

[0036] Inclisiran is a small interfering RNA (siRNA) drug that reduces LDL-C by lowering PCSK9 levels both inside and outside of cells. The dosing regimen of inclisiran, which involves an initial two doses followed by maintenance therapy every six months, helps to improve patient compliance.

[0037] In a third aspect, the present application further provides use of the pharmaceutical composition in the preparation of a medicament for treating tumors.

[0038] As a preferred embodiment, the ratio of the PCSK9 inhibitor to the oncolytic virus in the composition is 0.01-200 mg: 10 3 ~ 10 9 PFU, preferably, the ratio is 0.1-200 mg: 10 4 ~ 10 9 PFU; further preferably, the ratio is 0.1-100 mg: 10 5 ~ 10 9 PFU.

[0039] As a preferred embodiment, the tumor is a tumor that is not sensitive to the oncolytic virus.

[0040] As a preferred embodiment, the tumor is a solid tumor.

[0041] As a preferred embodiment, the solid tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, or gastric cancer.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] The present application finds that the PCSK9 inhibitor can increase the anti-tumor effect of the oncolytic virus, so as to improve the therapeutic effectiveness of the oncolytic virus as an anti-tumor drug. The animal experiments in the example part prove that the combination of VSV△51 virus and Alirocumab can significantly cause improvement of the tumor immune microenvironment, thereby significantly enhancing the inhibition of tumor cells.

[0044] In the embodiments of the present application, we combined alirocumab and VSV△51 virus to act on non-MSS and MSS colorectal cancer subcutaneous tumor mouse models, and surprisingly found that the combination of PCSK9 inhibitor alirocumab and VSV△51 virus significantly reduced the size and weight of tumors and significantly improved the recovery rate of mice. For MSS colorectal cancer (CT26, Colon26) modeling mice, the tumor volume and weight of the combination group mice were significantly reduced. For CT26 modeling mice, the complete remission rate of the combination group mice was 42.9%, while the remission rate of the VSV△51 group was only 14.3%. Compared with the anti-tumor effect of VSV△51 virus alone, the combination of alirocumab and VSV△51 significantly improved the oncolytic effect. For non-MSS colorectal cancer (MC38) modeling mice, the tumor volume and weight of the combination group mice were also significantly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Schematic diagram of the administration time arrangement of the alirocumab and VSV△51 virus combination treatment group for the MSS colorectal cancer CT26 animal model;

[0046] Figure 2 Schematic diagram of the final tumor volume comparison of the VSV△51 virus alone group, the alirocumab alone group, and the alirocumab and VSV△51 virus combination treatment group for the MSS colorectal cancer CT26 animal model;

[0047] Figure 3 Schematic diagram of the complete remission rate of the control group mice for the MSS colorectal cancer CT26 animal model;

[0048] Figure 4 Schematic diagram of the complete remission rate of the VSV△51 infection alone group mice for the MSS colorectal cancer CT26 animal model;

[0049] Figure 5 Schematic diagram of the complete remission rate of the alirocumab alone group mice for the MSS colorectal cancer CT26 animal model;

[0050] Figure 6 Schematic diagram of the complete remission rate of the alirocumab / VSV△51 combination group mice for the MSS colorectal cancer CT26 animal model;

[0051] Figure 7 Schematic diagram of the administration time arrangement of the alirocumab and VSV△51 virus combination treatment group for the MSS colorectal cancer Colon26 animal model;

[0052] Figure 8 Figure 6 is a schematic diagram showing the change in tumor volume over time for the control group, the VSVΔ51 virus alone group, the Alirocumab alone group, and the Alirocumab and VSVΔ51 virus combination group in the MSS colorectal cancer Colon26 animal model.

[0053] Figure 9 Figure 7 is a schematic diagram showing the comparison of the final tumor volume for the VSVΔ51 virus alone group, the Alirocumab alone group, and the Alirocumab and VSVΔ51 virus combination group in the MSS colorectal cancer Colon26 animal model.

[0054] Figure 10 Figure 8 is a schematic diagram showing the comparison of the final tumor weight for the VSVΔ51 virus alone group, the Alirocumab alone group, and the Alirocumab and VSVΔ51 virus combination group in the MSS colorectal cancer Colon26 animal model.

[0055] Figure 11 Figure 9 is a schematic diagram showing the administration time schedule for the Alirocumab and VSVΔ51 virus combination group in the non-MSS colorectal cancer MC38 animal model.

[0056] Figure 12 Figure 10 is a schematic diagram showing the change in tumor volume over time for the control group, the VSVΔ51 virus alone group, the Alirocumab alone group, and the Alirocumab and VSVΔ51 virus combination group in the non-MSS colorectal cancer MC38 animal model.

[0057] Figure 13 Figure 11 is a schematic diagram showing the comparison of the final tumor volume for the VSVΔ51 virus alone group, the Alirocumab alone group, and the Alirocumab and VSVΔ51 virus combination group in the non-MSS colorectal cancer MC38 animal model.

[0058] Figure 14 Figure 12 is a schematic diagram showing the comparison of the final tumor weight for the VSVΔ51 virus alone group, the Alirocumab alone group, and the Alirocumab and VSVΔ51 virus combination group in the non-MSS colorectal cancer MC38 animal model. DETAILED DESCRIPTION

[0059] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in conjunction with specific examples.

[0060] In the following examples, the VSVΔ51 virus is derived from a genetically engineered vesicular stomatitis virus (available by the method of this document Lawson ND, Stillman EA, Whitt MA, Rose JK. Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci U S A. 1995 May 9; 92(10): 4477-81. doi: 10.1073 / pnas.92.10.4477. Erratum in: Proc Natl Acad Sci U S A 1995 Sep 12; 92(19): 9009. PMID: 7753828; PMCID: PMC41967.); human colorectal cancer cell lines CT26, Colon26, MC38 are purchased from Pnuo Sai; the calculation method of complete remission rate of mouse transplanted tumor is: complete remission rate (%) = number of completely remitted mice / total number of mice x 100%.

[0061] Example 1

[0062] 1. Materials:

[0063] High-sugar DMEM medium, RPMI-1640 medium, VSVΔ51 virus, human colorectal cancer cell lines CT26, Colon26, MC38, 4-week-old female BALB / c mice, 4-week-old female C57BL / 6J mice.

[0064] 2. Methods:

[0065] 2.1 Preparation of tumor animal model:

[0066] This experiment adopts a random, single-blind design. 2x10 6 CT26 (mouse colon cancer cells), 3x10 6 Colon26 (mouse colon cancer cells) were injected into the dorsal subcutaneous of 4-week-old BALB / c mice to prepare MSS colorectal cancer CT26 animal models, MSS colorectal cancer Colon26 animal models; 8x10 5 MC38 cells (mouse colon cancer cells) were injected into the dorsal subcutaneous of 4-week-old C57BL / 6J mice to prepare non-MSS colorectal cancer MC38 animal models.

[0067] 2.2 Administration method:

[0068] CT26 model group: when the tumor size reaches 200mm 3Time groups, including the untreated control group, the Alirocumab alone group, the VSVΔ51 infection alone group and the Alirocumab / VSVΔ51 combination group. The Alirocumab alone group was injected intraperitoneally with 10 mg / kg / day, the VSVΔ51 infection alone group was injected intravenously with VSVΔ51 virus 3 x 10 7 PFU / time, and the Alirocumab / VSVΔ51 combination group was given the same dose of Alirocumab and VSVΔ51 virus in the same way. The three groups were injected with Alirocumab for 5 times continuously, and VSVΔ51 for 2 times continuously, 7 mice in each group.

[0069] Colon26 model group: when the tumor size reached 200 mm 3 Time groups, including the untreated control group, the Alirocumab alone group, the VSVΔ51 infection alone group and the Alirocumab / VSVΔ51 combination group. The Alirocumab alone group was injected intraperitoneally with 10 mg / kg / day, the VSVΔ51 infection alone group was injected intravenously with VSVΔ51 virus 3 x 10 7 PFU / time, and the Alirocumab / VSVΔ51 combination group was given the same dose of Alirocumab and VSVΔ51 virus in the same way. The three groups were injected with Alirocumab for 5 times continuously, and VSVΔ51 for 2 times continuously, 7 mice in each group.

[0070] MC38 model group: when the tumor size reached 100 mm 3 Time groups, including the untreated control group, the Alirocumab alone group, the VSVΔ51 infection alone group and the Alirocumab / VSVΔ51 combination group. The Alirocumab alone group was injected intraperitoneally with 10 mg / kg / day, the VSVΔ51 infection alone group was injected intravenously with VSVΔ51 virus 3 x 10 7 PFU / time, and the Alirocumab / VSVΔ51 combination group was given the same dose of Alirocumab and VSVΔ51 virus in the same way. The three groups were injected with Alirocumab for 5 times continuously, and VSVΔ51 for 2 times continuously, 7 mice in each group.

[0071] 2.3 Tumor measurement method:

[0072] The length and width of the tumor were measured every two days, and the volume of the tumor was calculated according to the formula (length x width 2) / 2. After measuring the tumor volume, One way ANOVA statistics was performed, * means P<0.05, ** means p<0.01, *** means P<0.001, **** means P<0.0001, _x0007_.

[0073] 3. Experimental results

[0074] Results as shown in Figures 1 to 14 , tumor volume determined by pathological anatomy showed that, compared with the control group, the Alirocumab group and the VSV△51 infection group alone could only cause slight reduction of tumor volume, while the Alirocumab / VSV△51 combination group (Combination) could cause significant reduction of tumor volume ( Figures 2 to 6 , Figure 8 , 9 , 12, 13), and significant weight loss ( Figure 10 , 14 ). The above results show that Alirocumab and VSV△51 have a synergistic effect and can significantly improve the treatment effect of tumors.

[0075] It should be noted that the present application also uses Evolocumab, Tafolecimab and other PCSK9 inhibitors, which also achieve the same or similar effect as Alirocumab, and will not be repeated here.

[0076] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not a limitation on the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A pharmaceutical composition for treating colon cancer, characterized by, The composition consists of an oncolytic virus and a PCSK9 inhibitor; The oncolytic virus is a vesicular stomatitis virus, and the vesicular stomatitis virus is a VSV△51 strain; The PCSK9 inhibitor is alirocumab.

2. The use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating colon cancer.

3. Use according to claim 2, wherein the compound is ###0002### The colon cancer is colon cancer that is not sensitive to an oncolytic virus.

Images