Application of nucleoside analogue CL-351 in preparation of medicine for regulating immune function

Abstract

The invention discloses an application of a nucleoside analogue CL-351 in preparation of a medicine for regulating an immune function. Compared with the prior art, the invention discloses the immunomodulatory activity of the CL-351 and the application of the CL-351 combined with the PD1 / PDL1 antibody as an immunomodulator composition. The invention finds that the CL-351 can up-regulate immune-related proteins MHC-I and PD-L1 on the surface of a cell membrane. Experimental research proves that the CL-351 serving as an immunomodulator has a remarkable inhibition effect on C57BL / 6J immune normal mouse loaded LLC tumors. Besides, the research also finds that compared with a single drug, the CL-351 and the PD-L1 antibody have a synergistic anti-tumor effect after being combined for use, and both the CL-351 single drug group and the CL-351 combined PD-L1 antibody group can obviously change the immune cell infiltration condition in tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of new drug uses, and particularly relates to the application of the nucleoside analog CL-351 in the preparation of a drug for regulating immune function. Background Art

[0002] Patent document CN 119033801 A discloses the pharmaceutical uses of 1-(4-alkynyl-2-deoxy-2-fluoro-β-D-ribofuranosyl) cytosine (CL-351) or its salts. The nucleoside analog CL-351 can be used for treating tumors, such as leukemia, lymphoma, pancreatic cancer, lung cancer, gastric cancer, liver cancer, breast cancer, etc. It is found that the nucleoside analog CL-351 has obvious inhibitory effects on a variety of human cancer cells and transplanted tumors in animals. The structure of the nucleoside analog CL-351 is shown as follows:

[0003]

[0004] The pharmaceutical uses of CL-351 are worthy of further expansion. Summary of the Invention

[0005] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides the application of the nucleoside analog CL-351 in the preparation of a drug for regulating immune function. On the basis of the prior art, the present invention further studies the nucleoside analog CL-351 and finds that in addition to its function as an anti-tumor drug, CL-351 can also have good immune regulatory effects, can be used as an immune regulator, and after being combined with PD-L1 / PD-1 antibodies, has a significant synergistic effect and significantly better immune regulatory activity.

[0006] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides the application of the nucleoside analog CL-351, or its pharmaceutically acceptable salt, stereoisomer or isotope derivative in the preparation of a drug for regulating immune function.

[0008] In the second aspect, the present invention provides the application of the nucleoside analog CL-351, or its pharmaceutically acceptable salt, stereoisomer or isotope derivative in the preparation of a drug for treating immune-related diseases.

[0009] As a specific embodiment, the immune-related diseases are viral infections or tumors.

[0010] In the third aspect, the present invention provides the application of the nucleoside analog CL-351, or its pharmaceutically acceptable salt, stereoisomer or isotope derivative, in combination with PD1 / PDL1 antibodies in the preparation of a drug for regulating immune function.

[0011] Fourth aspect, the present invention provides the use of nucleoside analogue CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof, in combination with a PD1 / PDL1 antibody in the preparation of a medicament for treating immune-related diseases.

[0012] As a specific embodiment, the immune-related disease is a viral infection or a tumor.

[0013] Fifth aspect, the present invention provides a pharmaceutical composition comprising nucleoside analogue CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof, wherein the nucleoside analogue CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof, is used as an immunomodulatory active ingredient.

[0014] As a specific embodiment, the pharmaceutical composition further comprises a PD1 / PDL1 antibody as an immunomodulatory active ingredient.

[0015] Sixth aspect, the present invention provides the use of the pharmaceutical composition as described above in the preparation of a medicament for regulating immune function.

[0016] Seventh aspect, the present invention provides the use of the pharmaceutical composition as described above in the preparation of a medicament for treating immune-related diseases.

[0017] As a specific embodiment, the immune-related disease is a viral infection or a tumor.

[0018] In a preferred embodiment of the present invention, the above-mentioned PD-1 antibody is selected from any one or a combination of pembrolizumab, nivolumab, sintilimab, toripalimab, RMP1-14, camrelizumab, tislelizumab and cemiplimab, etc.

[0019] In a preferred embodiment of the present invention, the above-mentioned PD-L1 antibody is selected from any one or a combination of avelumab, atezolizumab and durvalumab, etc.

[0020] In a preferred embodiment of the present invention, the above-mentioned PD-1 antibody is selected from humanized antibodies having complementary determining regions identical to those of the RMP1-14 antibody.

[0021] The present invention also provides a method for regulating or enhancing an immune response in vivo, comprising: administering an effective amount of CL-351 to an individual in need thereof.

[0022] In another aspect, the present invention also provides a method for enhancing immune response, comprising: obtaining a sample from a patient; isolating immune cells from the sample; culturing the immune cells with CL-351; amplifying the immune cells and reintroducing the cells into the patient; and enhancing the immune response.

[0023] In a preferred embodiment of the present invention, the immune cells include but are not limited to antigen-presenting cells, T cells, B cells, natural killer cells, etc.

[0024] In a preferred embodiment of the present invention, it further includes a second immunologically active substance.

[0025] In a preferred embodiment of the present invention, the second immunologically active substance is a PD1 / PDL1 antibody or a combination thereof.

[0026] In a preferred embodiment of the present invention, the PD-1 antibody is selected from any one or a combination of pembrolizumab, nivolumab, sintilimab, toripalimab, RMP1-14, camrelizumab, tislelizumab, and cemiplimab, etc.

[0027] In a preferred embodiment of the present invention, the PD-L1 antibody is selected from any one or a combination of avelumab, atezolizumab, and durvalumab, etc.

[0028] On the other hand, the present invention also provides the use of CL-351 in enhancing the infiltration and proliferation of immune cells.

[0029] In a preferred embodiment of the present invention, the immune cells include but are not limited to NK cells, CD8+ T cells, CD4+ T cells, eosinophils, and mast cells.

[0030] Beneficial effects: Compared with the prior art, the present invention discloses the immunomodulatory activity of CL-351 and the use of CL-351 in combination with PD1 / PDL1 antibodies as an immunomodulatory agent composition. The present invention finds that CL-351 can upregulate the immune-related proteins MHC-I and PD-L1 on the cell membrane surface. Experimental studies of the present invention confirm that CL-351, as an immunomodulator, has a significant inhibitory effect on LLC tumor-bearing C57BL / 6J immunocompetent mice. In addition, the present invention also finds that, compared with single drug, the combination of CL-351 and PD-L1 antibody has a synergistic anti-tumor effect, and both the CL-351 single drug group and the CL-351 combined with PD-L1 antibody group can significantly change the infiltration of immune cells in tumors. Description of the Drawings

[0031] Figure 1Compound CL-351 upregulates the expression level of MHC-I in SU-DHL-4 cells.

[0032] Figure 2 Compound CL-351 upregulates the expression level of PD-L1 in BxPC3 cells.

[0033] Figure 3 Effect of single-agent CL-351 and combination administration with PD-L1 antibody on tumor volume in a murine model of LLC-bearing lung cancer.

[0034] Figure 4 Effect of single-agent CL-351 and combination administration with PD-L1 antibody on the distribution difference of major subsets of immune cells in tumors of a murine model of LLC-bearing lung cancer.

[0035] Figure 5 Histogram of the effect of single-agent CL-351 and combination administration with PD-L1 antibody on the distribution difference of major subsets of immune cells in tumors of a murine model of LLC-bearing lung cancer. Detailed implementation manners

[0036] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0037] Example 1

[0038] 1. Experimental materials

[0039] CL-351, off-white powder, purity greater than 98%, relative molecular mass 269, provided by the School of Pharmacy, Zhengzhou University; RPMI 1640 complete medium, produced by Gibco; fetal bovine serum, produced by Gibco; penicillin-streptomycin, produced by Gibco; trypsin, produced by Gibco; PD-L1 antibody for flow cytometry, produced by Biolegend; APC anti-human HLA-A / B / C antibody for flow cytometry, produced by Biolegend; PBS (pH = 7.4) solution, etc.

[0040] Cell lines:

[0041]

[0042] 2. Instrument and equipment

[0043]

[0044] 3. Experimental methods

[0045] 3.1 Flow cytometry - Detection of the expression level of PD-L1 on the surface of BxPC3 cells

[0046] a) First, collect the cultured BxPC3 cells into a single-cell suspension and count them using a hemocytometer.

[0047] b) Use a multi-channel pipette to add samples to a 96-well transparent flat-bottom plate, adding 100 μL to each well to ensure that the cell count per well is 1.5×10 4 cells. Then place the cells in a carbon dioxide cell incubator for overnight culture.

[0048] c) After the cells have adhered overnight, quickly aspirate the culture medium supernatant using a vacuum pump and add drugs using a multi-channel pipette. After adding the drugs, return the cells to the carbon dioxide incubator for culture.

[0049] e) After 48 hours of compound treatment, take out the cell culture plate, quickly flick off the culture medium supernatant, and use a multi-channel pipette to aspirate 200 μL of PBS for washing. After washing, flick off the PBS and invert the plate on filter paper and gently tap to quickly dry the residual liquid in the plate.

[0050] f) Use a multi-channel pipette to aspirate 20 μL of trypsin solution into the cell culture plate and place it in a 37°C incubator for digestion.

[0051] g) Prepare an anti-PD-L1 antibody solution in complete medium at a ratio of 1:3000. After the cells in the cell culture plate are digested, use a multi-channel pipette to aspirate 30 μL of the prepared anti-PD-L1 antibody solution for neutralization, and use a multi-channel pipette to pipette and mix the cells until they are in a single-cell state. The final dilution ratio of the anti-PD-L1 antibody is 1:5000.

[0052] h) After staining in the dark at 4°C for 30 minutes, use a high-throughput flow cytometer IntelliCyt iQue ScreenerPLUS to detect the expression of PD-L1 on the surface of BxPC3 cells.

[0053] 3.2 Flow cytometry - Detection of MHC-I expression level on the surface of SU-DHL-4 cells

[0054] a) Dilute the cultured SU-DHL-4 cells with complete medium to 7.5×10 4 cells / mL.

[0055] b) Use a multi-channel pipette to add the cell suspension to a 96-well transparent U-bottom plate, with 1.5×10 4 cells / 200 μL in each well. Then place the cells in a carbon dioxide cell incubator for overnight culture.

[0056] c) Use a multi-channel pipette to add drugs.

[0057] d) After the drug addition is completed, return the cells to the cell incubator and culture for 48 hours.

[0058] e) Take out the 96-well plate, and use a multi-channel pipette to aspirate 160 μL of the culture medium supernatant. Prepare an APC anti-human HLA-A / B / C antibody solution with complete medium at a ratio of 1:200, and use a multi-channel pipette to aspirate 10 μL of the prepared antibody solution (antibody dilution ratio is 1:1000) and add it to the 96-well plate.

[0059] f) Incubate in the dark at 4 °C for 30 minutes, and detect using a high-throughput flow cytometer IntelliCyt iQue ScreenerPLUS.

[0060] 4. Experimental Results

[0061] 4.1 Results of compound CL-351 upregulating the expression level of MHC-I

[0062] After treating SU-DHL-4 cells with 40 / 10 / 2.5 / 0.63 / 0.16 / 0.04 / 0.01 / 0.002 / 0.0006 μM of CL-351 for 48 h, incubate with anti-human HLA-A / B / C and then detect the expression of MHC-I on the cell surface by flow cytometry. As Figure 1 shown, CL-351 can increase the expression level of MHC-I (HLA-A / B / C) on the cell membrane surface of SU-DHL-4 in a concentration-dependent manner, with an EC 50 of 4.941 μM, and upregulating the expression of MHC-I by about 2.0-fold at 40 μM.

[0063] 4.2 Results of compound CL-351 upregulating the expression level of PD-L1

[0064] After treating BxPC3 cells with 40 / 10 / 2.5 / 0.63 / 0.16 / 0.04 / 0.01 / 0.002 / 0.0006 μM of CL-351 for 48 h, incubate with anti-human PD-L1 and then detect the expression of PD-L1 on the cell surface by flow cytometry. As Figure 2 shown, CL351 can increase the expression level of PD-L1 on the cell membrane surface of BxPC3 in a concentration-dependent manner, with an EC 50 of 694 nM, and CL-351 upregulating the expression of PD-L1 by about 2.6-fold at 40 μM.

[0065] Example 2. Effects of the CL-351 single-drug group and the combined use of the PD-L1 antibody group on anti-tumor in normal immune mice, including the effects on the tumor growth inhibition rate and the immune microenvironment in the tumor

[0066] 1. Experimental Materials, Experimental Animals and Breeding Environment

[0067] Experimental animals: C57BL / 6J mice, 6 - 8 weeks old, weighing 18.0 - 22.5 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0068] Experimental materials: CL-351, off-white powder, purity greater than 98%, relative molecular mass of 269, provided by the School of Pharmacy, Zhengzhou University; InVivoMab anti-mouse PD-L1, liquid, purity greater than 95%, purchased from Bioxcell

[0069] 2. Cell Culture

[0070] LLC cells, cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO 2 incubator. Routine subculture was performed once a week. When the cell confluence reached 80% - 90% and the cell number met the requirements, the cells were harvested, counted, and seeded.

[0071] 3. Tumor Cell Inoculation

[0072] LLC cells were subcutaneously inoculated into each mouse to form a subcutaneous bump the size of a soybean.

[0073] 4. Grouping and Treatment of Mouse Tumor Xenograft Models

[0074] When the tumor volume reached about 100 mm 3 the mice were randomly divided into 4 groups of 6 mice each. The corresponding treatment regimens were as follows:

[0075] (1) Control group: Administered intraperitoneal injection of sterile ultrapure water daily;

[0076] (2) αPD-L1 monotherapy group (αPD-L1): Administered αPD-L1 at a dose of 10 mg / kg intraperitoneally twice a week;

[0077] (3) CL-351 monotherapy group: Administered CL-351 at a dose of 1 mg / kg intraperitoneally daily;

[0078] (4) Combination group: Administered CL-351 at a dose of 1 mg / kg intraperitoneally daily and αPD-L1 at a dose of 10 mg / kg intraperitoneally twice a week for 10 consecutive days.

[0079] Based on the daily monitoring data, a tumor growth curve was plotted, and the tumor tissues were dissected 10 days after drug administration.

[0080] The experimental indicators are to examine whether tumor growth is inhibited, delayed or cured. The tumor diameter is measured with vernier calipers three times a week.

[0081] The formula for calculating the tumor volume is: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor respectively.

[0082] The antitumor efficacy of the compound is evaluated by TGI (%). TGI (%) can reflect the tumor growth inhibition rate. The calculation of TGI (%): TGI (%) = 【1 - (the average tumor volume at the end of drug administration in a certain treatment group - the average tumor volume at the start of drug administration in this treatment group) / (the average tumor volume at the end of treatment in the solvent control group - the average tumor volume at the start of treatment in the solvent control group)】× 100%.

[0083] 5. Experimental results

[0084] The results of the tumor growth curve test are shown in the appendix Figure 3 . It can be seen from the test results that both the CL-351 single-drug group and the dual-drug combination group have obvious antitumor effects on the C57BL / 6J immunocompetent mouse bearing LLC tumor model, with TGI being 82.7% and 92.6% respectively, and the antitumor effect is enhanced after CL-351 is combined with αPD-L1. CL-351 combined with αPD-L1 can improve the antitumor effect of the single drug αPD-L1 in this LLC cell line lung cancer, and the TGI is increased from 25.0% to 92.6%.

[0085] The body weight of the experimental animals is used as a reference index for indirectly measuring drug toxicity. The tumor-bearing mice showed good tolerance to different doses of the test drugs, and there was no obvious weight loss in all treatment groups.

[0086] The tumor samples were sent to Zhejiang Proting Health Technology Co., Ltd. for further study on the immune microenvironment in 4 groups of tumors, and mass cytometry (CyTOF) was used for detection and analysis.

[0087] Mass cytometry is a protein expression profiling technology at the single-cell level, which is a high-throughput flow cytometry technology using metal isotopes instead of traditional fluorescent labels.

[0088] The comparison of the distribution differences of the major subsets of immune cells in the above 4 groups of experimental tumors is shown in the appendix Figure 4 and appendix Figure 5 , and it can be seen from the results that after administration in the CL-351 single-drug group and the dual-drug combination group, NK cells, CD4 + T, CD8 +Increased infiltration of T cells, eosinophils, and mast cells, which was more significant in the dual-drug combination group than in the CL-351 monotherapy group; the proportion of myeloid cells was significantly reduced, mainly due to the significant reduction in the proportions of M2 macrophages and M-MDSC cells.

[0089] Based on this experiment, CL-351 can promote the infiltration and proliferation of NK cells, CD4 + T cells, CD8 + T cells, eosinophils, and mast cells to exert an anti-tumor effect, further demonstrating that CL-351 can be used as an immunomodulator.

[0090] Based on this experiment, the dual-drug combination group can promote the infiltration and proliferation of NK cells, CD8 + T cells, CD4 + T cells, eosinophils, and mast cells to exert an anti-tumor effect, and has a stronger immunomodulatory effect than CL-351 monotherapy, further demonstrating that the combination of CL-351 and the PD-L1 antibody can be used as an immunomodulatory combination agent.

[0091] The embodiments of the present invention have been described in detail above in conjunction with specific embodiments. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art.

Claims

1. Use of the nucleoside analogue CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof in the preparation of a drug for regulating immune function.

2. Use of the nucleoside analogue CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof in the preparation of a drug for treating immune-related diseases.

3. The use according to claim 2, characterized in that: The immune-related disease is a viral infection or a tumor.

4. Use of the nucleoside analog CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof, in combination with a PD1 / PDL1 antibody in the preparation of a drug for regulating immune function.

5. Use of the nucleoside analog CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof, in combination with a PD1 / PDL1 antibody in the preparation of a medicament for the treatment of immune-related diseases.

6. The use according to claim 5, characterized in that: The immune-related disease is a viral infection or a tumor.

7. A pharmaceutical composition comprising a nucleoside analog CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof, characterized in that: In the pharmaceutical composition, the nucleoside analog CL-351, or a pharmaceutically acceptable salt, stereoisomer or isotope derivative thereof is used as an immunomodulatory active ingredient.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also includes PD1 / PDL1 antibody as an immunomodulatory active ingredient.

9. Use of the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for regulating immune function.

10. Use of the pharmaceutical composition according to claim 7 or 8 in the preparation of a medicament for treating an immune-related disease; preferably, the immune-related disease is a viral infection or a tumor.

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