Medicine compound and application thereof in preparation of medicine for treating tumors

Abstract

The invention provides a compound for treating tumors and application thereof. The compound comprises an immunomodulatory drug and a PD1 antibody. The immunomodulatory drugs involved in the invention are respectively a hybrid molecule GD1 of gemcitabine and dexamethasone, a dexamethasone precursor Pro-DEX, and a recombinant fusion protein DF-HSA of human beta defensin and human serum albumin. The immunomodulatory drug and the PD1 antibody in the compound have a remarkable synergistic effect, and the compound has the advantages of improving the anti-tumor defects of the PD1 antibody, targeting a tumor area, synergistically improving the anti-tumor efficiency, inhibiting the activity of tumor cells, weakening the drug resistance of the tumor cells to chemotherapeutic drugs, slowing down the recurrence and metastasis of the tumor cells and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of a tumor treating compound in the field of anti-tumor. Background Art

[0002] The morbidity and mortality of tumors are major health problems worldwide. In recent years, the morbidity and mortality of malignant tumors in my country have continued to rise. Traditional treatments for tumors have limited efficacy and urgently need the development of innovative ideas. Tumor immunotherapy has ushered in a new era of solid tumor treatment. Its principle is to combine immunology with emerging tumor treatment methods to improve the immunogenicity of tumor cells and their sensitivity to effector cell killing, relieve the body's inhibitory effects on immune cells, and reactivate immune cells, thereby clearing tumor cells and inhibiting tumor growth. However, because tumors have a variety of immune escape mechanisms to evade the monitoring and elimination of the immune system, the immunotherapy effect of most patients is still not ideal. In recent years, immune checkpoint blockade drugs represented by PD1 / PD-L1 have achieved unprecedented results in the field of tumor treatment.

[0003] Programmed Death 1 (PD1) is a cell surface receptor that inhibits T cell inflammatory activity. It is a member of the B7-CD28 receptor family and is often expressed in patients' T cells. Programmed cell death-ligand 1 (PD-L1) is a co-stimulatory signal molecule in the B7 family and is expressed in a variety of tumor cells and tumor tissues. Tumor cells express PD-L1 on the surface and bind to PD-1 on the surface of T cells to produce inhibitory signals, affecting T cell function and thus evading the attack of the immune system. The PD1 / PD-L1 pathway plays an important role in inducing effector T cell apoptosis, inhibiting T cell activation, inhibiting the body's anti-tumor immune response and tumor immune escape. At present, the immune checkpoint PD1 / PD-L1, as an important component marker involved in the body's tumor immune regulation process and a research hotspot for tumor immunotherapy, has been successfully applied to the clinical treatment of malignant tumors such as melanoma and breast cancer. Studies have shown that in unselected advanced cancer patients, the efficacy of PD1 antibodies alone is only 10%-30%. This result causes PD1 antibodies to have low clinical effective response rates and unclear optimal beneficiary populations. At present, studies at home and abroad have shown that when PD1 antibodies are used in combination with other anti-tumor drugs, they have a good effect on the treatment of malignant tumors. The combination of drugs can significantly improve the defects of PD1 antibodies and play a synergistic role in enhancing the anti-tumor effect.

[0004] In recent years, genetic engineering fusion proteins have been widely used in the fields of biotechnology and medical research. After the fusion genes are expressed, a new type of multi-domain artificially synthesized protein with multiple functions can be obtained by splicing together different functional proteins. In the early stage, the inventors of the present invention prepared the recombinant fusion protein DF2-HSA of defensin and human serum albumin (Chinese invention patent application CN 117338906A). Studies have shown that DF2-HSA can inhibit the proliferation, migration and invasion of tumor cells, weaken their stem cell characteristics, and can be specifically distributed in the tumor site, and has great potential for the treatment of solid tumors.

[0005] Dexamethasone is a synthetic glucocorticoid with antiviral, anti-rheumatic, anti-allergic and anti-inflammatory effects. It is mainly used clinically for allergic and autoimmune diseases, such as skin diseases, allergic reactions, asthma, etc. The latest research has shown that dexamethasone can assist drugs in anti-tumor effects by regulating cytokines, and can also be used as a sensitizer for anti-tumor drugs, showing great potential in tumors. The dexamethasone prodrug Pro-DEX (i.e., the compound of formula I or formula II or its pharmaceutical salt prepared according to Chinese invention patent CN 115636863B) prepared by the inventor of the present invention in the early stage can bind to albumin in the blood and be released in the acidic environment of the tumor to achieve the effect of endogenous ADC (Antibody-Drug Conjugates) drugs.

[0006] Gemcitabine is a commonly used anti-solid tumor drug in clinical practice, with a wide anti-tumor spectrum, and is a new generation of deoxycytidine nucleoside analogs. However, gemcitabine is prone to drug resistance and potential toxicity to normal tissues, and the administration method is injection administration, and patient compliance is poor. The staff of the department hopes to transform it to obtain a new structure with high efficacy, strong safety, and high patient compliance. The inventors previously invented a hybrid molecule GD1 of gemcitabine and dexamethasone (i.e., a compound of formula I, formula II or formula III prepared according to Chinese invention patent CN113307838B). The experimental results show that GD1 can overcome the drug resistance of gemcitabine, improve the therapeutic efficacy, improve the route of administration, and reduce toxic side effects. Summary of the invention

[0007] In view of the low clinical effective response rate and unclear optimal beneficiary population of PD1 antibodies in tumor immunotherapy, the present invention provides a new anti-tumor drug complex to overcome the defects of existing PD1 antibodies and meet clinical needs.

[0008] In order to achieve the above-mentioned object, the present invention provides a drug complex, which includes an immunomodulatory drug and a PD1 antibody, wherein the immunomodulatory drug is selected from the recombinant genetic engineering fusion protein DF2-HSA of defensin and human serum albumin, the dexamethasone prodrug Pro-DEX and the hybrid molecule GD1 of gemcitabine and dexamethasone.

[0009] In the present invention, the recombinant genetic engineering fusion protein DF2-HSA of defensin and human serum albumin also includes pharmaceutically acceptable derivatives thereof. Those skilled in the art can prepare it according to the method described in Chinese invention patent application CN 117338906A.

[0010] Those skilled in the art can prepare the compound of formula I or formula II or its pharmaceutically acceptable salt according to the description of Chinese invention patent CN 115636863B as the dexamethasone prodrug Pro-DEX of the present invention; and can prepare the compound of formula I, formula II or formula III as the hybrid molecule GD1 of gemcitabine and dexamethasone of the present invention according to the description of Chinese invention patent CN113307838B.

[0011] The present invention also provides the use of the drug complex in preparing drugs for treating tumors.

[0012] Wherein, the tumor is breast cancer, lung cancer, esophageal cancer, pancreatic cancer, non-small cell carcinoma, gastric cancer, liver cancer, colon cancer, prostate cancer, cervical cancer and / or rectal cancer.

[0013] In the present invention, the mass concentration of the PD1 antibody to tumor cells is 0.1 mg / Kg to 500 mg / Kg, preferably 10 mg / Kg; the mass concentration of the immunomodulatory drug to tumor cells is 0.1 mg / Kg to 500 mg / Kg, preferably 10 mg / Kg.

[0014] The present invention uses a mouse breast cancer transplant tumor model to prove that the drug complex of the present invention can significantly inhibit tumor growth and show a synergistic effect. Compared with the use of PD1 antibody alone, the complex of the present invention enhances the anti-tumor effect of PD1 antibody, effectively improves the therapeutic effect of PD1 antibody, and shows great potential in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The growth inhibition effect of the complex on mouse breast cancer 4T1-luc (tumor volume);

[0016] Figure 2 Effect of the complex on body weight of mice bearing 4T1-luc tumors of mouse breast cancer;

[0017] Figure 3Growth inhibitory effect (tumor weight) of the complex on murine breast cancer 4T1-luc. Detailed implementation mode

[0018] The following listed examples are only to help those skilled in the art better understand the present invention, but do not limit the present invention in any way.

[0019] In the present invention, unless otherwise specified, "%" for explaining concentration is weight percentage, and ":" is weight ratio.

[0020] In the present invention, the general formula compounds of the hybrid molecule GD1 prepared according to Chinese invention patent CN 113307838B all contain gemcitabine and dexamethasone active groups, and the present inventors have confirmed that there is no obvious difference in the anti-tumor activity of different compounds. Therefore, the general formula compounds have the same pharmacological activity and they are equivalent.

[0021] In the present invention, the general formula compound Pro-DEX prepared according to Chinese invention patent CN 115636863B is dexamethasone with maleimide fragments containing different linkers, and the present inventors have confirmed that there is no obvious difference in the anti-tumor activity of different compounds. Therefore, the general formula compounds have the same pharmacological activity and they are equivalent.

[0022] The present invention relates to the following drugs, reagents and experimental animals:

[0023] Gemcitabine hydrochloride raw material (purity 98%) was purchased from Zhengzhou Alpha Chemical Co., Ltd.; dexamethasone raw material (purity 98%) was purchased from Shanghai Xinkai Chemical Technology Co., Ltd.; hybrid molecule GD1 (compound 1 prepared according to Chinese invention patent CN113307838B), Pro-DEX (compound 2 prepared according to Chinese invention patent CN 115636863B), DF2-HSA (prepared according to Chinese invention patent application CN 117338906A) are from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences; murine PD1 antibody was purchased from Abcam; murine breast cancer cell line 4T1-luc cells were purchased from Shanghai Core Ray Biotechnology Co., Ltd.

[0024] Healthy female BALB / c mice aged 4 - 6 weeks, 18 - 22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number [SCIK(Beijing)2022 - 0010]).

[0025] Example 1 In vivo anti-tumor activity study of the drug - inhibitory effect on murine tumor growth

[0026] Dilute the 4T1-luc cells in the logarithmic growth phase to 1×10 7 / ml cell suspension, 0.1ml / mouse was inoculated into the subcutaneous part of the armpit of BALB / c mice. After the tumor mass grew to a certain volume, the tumor mass was removed under sterile conditions and cut into 2mm 3 Small pieces of uniform size were inoculated subcutaneously in the axilla of BALB / c mice using a trocar. On the third day, the mice were divided into 8 groups, 6 in each group, namely: control group, PD1 antibody group, GD1 group, Pro-DEX group, DF2-HSA group, PD1 antibody + GD1 group, PD1 antibody + Pro-DEX group, and PD1 antibody + DF2-HSA group.

[0027] Among them, PD1 antibody was injected intraperitoneally, once every 3 days for a total of 4 times; GD1 was orally administered, once a day for a total of 10 times; Pro-DEX and DF2-HSA were both injected into the tail vein, once every 3 days for a total of 4 times. The dosing schedule is shown in Table 1.

[0028] Table 1 Dosage regimen for mouse breast cancer 4T1-luc subcutaneous transplant tumor model

[0029]

[0030] The tumor size and mouse body weight were measured on the 5th, 7th, 9th and 11th day after administration, and the tumor volume change was calculated. The tumor mass was removed and weighed on the 14th day after inoculation, and the tumor mass volume was calculated.

[0031] Tumor volume calculation formula V = ab 2 / 2 (a: long diameter of tumor, b: short diameter of tumor).

[0032] The growth inhibition effect of the complex provided by the present invention on mouse breast cancer 4T1-luc was investigated by tumor volume. The results are as follows Figure 1 When used alone, PD1 antibody has little inhibitory effect on tumors. When combined with immunomodulatory drugs, it has a significant inhibitory effect on tumor growth. The inhibitory effect of the complex composed of PD1 antibody and DF2-HSA is the most significant.

[0033] On the other hand, the effect of the complex of the present invention on the body weight of 4T1-luc cell-bearing mice is as follows: Figure 2 The results showed that the average body weight of mice in the control group, PD1 antibody group, GD1 group, Pro-DEX group, DF2-HSA group, PD1 antibody + GD1 group, PD1 antibody + Pro-DEX group, and PD1 antibody + DF2-HSA group did not show significant differences from the initial body weight, indicating that the complex of the present invention has strong safety for mice.

[0034] Furthermore, the tumor inhibition rate was calculated based on the weight of the subcutaneous tumors in each group at the end of the experiment, and the data were statistically analyzed.

[0035] Tumor inhibition rate = (average tumor weight of the control group - average tumor weight of the experimental group) / average tumor weight of the control group × 100%

[0036] According to the calculation formula of the coefficient of drug interaction (CDI): CDI = AB / (A×B), AB is the ratio of tumor weight between the two-drug combination group and the control group, and A or B is the ratio of tumor weight between the drug single-drug group and the control group. When CDI < 1, it indicates that the two drugs act synergistically, when CDI < 0.7, it indicates that the two drugs have a very significant synergistic effect, when CDI = 1, it indicates that the two drugs are additive, and when CDI > 1, the two drugs act antagonistically. A t test was performed at the same time.

[0037] The growth inhibition effect of the complex of the present invention on mouse breast cancer 4T1-luc was investigated by tumor weight and tumor inhibition rate. Figure 3 And Table 2.

[0038] Table 2 Effects of drugs on subcutaneous transplanted tumors of breast cancer 4T1-luc in mice

[0039]

[0040] *P<0.05vs.PD1, **P<0.01vs.PD1, ***P<0.001vs.PD1

[0041] The results of tumor inhibition rate calculation showed that the tumor inhibition effect of PD1 antibody alone was poor, the tumor inhibition rate of PD1 antibody combined with GD1 was 60.58%, the tumor inhibition rate of PD1 antibody combined with Pro-DEX was 58.15%, and the tumor inhibition rate of PD1 antibody combined with DF2-HSA was 75.18%. The complex composed of PD1 antibody and DF2-HSA had the most significant tumor inhibition effect. The results of CDI calculation showed that the interaction coefficient between PD1 antibody and GD1 was 0.74, the interaction coefficient between PD1 antibody and Pro-DEX was 0.71, and the interaction coefficient between PD1 antibody and DF2-HSA was 0.61.

[0042] The above results show that PD1 antibody is insensitive to mouse breast cancer 4T1-luc transplanted tumors. After forming a complex with three immunomodulatory drugs, the tumor growth inhibition effect is significantly improved compared with the single drug group, and the CDI is less than 1, showing a synergistic effect. Among them, the synergistic effect of PD1 antibody + DF2-HSA is the most significant.

Claims

1. A drug complex, comprising a PD1 antibody and an immunomodulatory drug, wherein the immunomodulatory drug is selected from a recombinant genetic engineering fusion protein DF2-HSA of defensin and human serum albumin, a dexamethasone prodrug Pro-DEX, and a hybrid molecule GD1 of gemcitabine and dexamethasone.

2. The drug complex according to claim 1, characterized in that The recombinant genetic engineering fusion protein DF2-HSA of defensin and human serum albumin includes pharmaceutically acceptable derivatives thereof.

3. The composite according to claim 1, characterized in that The structure of the dexamethasone prodrug Pro-DEX is a compound of formula I or formula II or a pharmaceutically acceptable salt thereof prepared according to Chinese invention patent application CN 115636863B.

4. The composite according to claim 1, characterized in that The structure of the hybrid molecule GD1 of gemcitabine and dexamethasone is the compound of formula I, formula II or formula III prepared according to Chinese invention patent application CN 113307838B.

5. Use of the drug complex according to claims 1-4 in preparing drugs for treating tumors.

6. The use according to claim 5, characterized in that The tumor is breast cancer, lung cancer, esophageal cancer, pancreatic cancer, non-small cell carcinoma, stomach cancer, liver cancer, colon cancer, prostate cancer, cervical cancer and / or rectal cancer.

7. The use according to claim 5, characterized in that The mass concentration of the PD1 antibody to tumor cells is 0.1 mg / Kg to 500 mg / Kg, and the mass concentration of the immunomodulatory drug to tumor cells is 0.1 mg / Kg to 500 mg / Kg.

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