CN106539811B - Application of cyclic dinucleotide cGAMP in preventing and treating complications induced by antineoplastic drugs or reducing toxic and side effects induced by chemotherapeutic drugs - Google Patents

Abstract

The invention belongs to the field of medicine technology and tumor immunotherapy, and particularly relates to application of cyclic dinucleotide (cGAMP) in preventing and treating complications of antitumor chemotherapy and reducing toxic and side effects of chemotherapeutics. The research of the invention shows that the cGAMP can prevent and treat complications induced by the antitumor chemotherapeutic drugs, enhance the curative effect of the antitumor chemotherapeutic drugs and reduce the toxicity of the chemotherapeutic drugs. The mouse tumor model shows that the cGAMP and the 5-fluorouracil can enhance the effect of the 5-fluorouracil on inhibiting the colorectal cancer cell MC38 and can reduce the intestinal mucosa injury induced by using the 5-fluorouracil; the cGAMP combined cyclophosphamide can enhance and inhibit lymphocyte leukemia L1210, prolong the survival cycle of mice, increase the number of erythrocytes, platelets and neutrophils in blood, and has the effects of preventing and treating complications such as anemia and low immunity caused by using an anti-leukemia chemotherapeutic drug. Therefore, the cyclic dinucleotide cGAMP can be used together with an anti-tumor medicament to prevent and treat complications caused by chemotherapeutic medicaments or reduce toxic and side effects caused by chemotherapy.

Description

Application of cyclic dinucleotide cGAMP in preventing and treating complications induced by antineoplastic drugs or reducing toxic and side effects induced by chemotherapeutic drugs

Technical Field

The invention belongs to the fields of biological medicine technology and tumor immunotherapy, and particularly relates to application of cyclic dinucleotide (cGAMP) and derivatives thereof in combined therapy of tumors and reduction of toxicity of antitumor drugs.

Background

Tumors are one of the major diseases seriously harming human life and health, and are manifested by abnormal cell hyperproliferation and differentiation. The WHO experts forecast that the tumor incidence of the global population will reach 2000 million people and the death number will reach 1200 million people in 2020, and the tumor will become the first killer of the human in the century and form the most serious threat to the human survival. The morbidity and mortality of lung cancer, colorectal cancer, gastric cancer, liver cancer and the like are in the prostate of various malignant tumors. According to statistics of (2012 annual report of Chinese tumor registration) issued by national tumor registration center, about 312 million new tumor cases occur every year, 8550 people are in average every day, and 6 people are diagnosed as cancer every minute in the whole country.

5-fluorouracil, abbreviated as 5-FU, 5-fluorouracil is a pyrimidine fluoride, belongs to an antimetabolite antitumor drug, and can inhibit thymidylate synthase, block the conversion of deoxypyrimidine nucleotide into thymidine nucleus, and interfere with DNA synthesis. Also has certain inhibition effect on RNA synthesis. The composition is clinically used for colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, chorioepithelioma, malignant hydatidiform mole, head and neck squamous carcinoma, skin cancer, liver cancer, bladder cancer and the like. 5-FU is the first antimetabolite synthesized according to certain assumption, is the most widely used anti-pyrimidine medicine in clinic, has good curative effect on digestive tract cancer and other solid tumors, and plays an important role in medical treatment of tumors. 5-fluorouracil is converted into 5-fluorodeoxyuridine by enzyme to have antitumor activity. 5-FU inhibits DNA synthesis by inhibiting thymidylate synthase. The action of this enzyme may transfer one carbon unit of formyltetrahydrofolate to deoxyuridine-phosphate to synthesize thymidine monophosphate. 5-FU also has a certain inhibitory effect on RNA synthesis. 5-FU has cytotoxicity after undergoing biotransformation effects such as ribosylation and phosphorylation in vivo. 5-FU can generate F-dUMP and FUMP via different pathways. The former can be covalently bound to the active center of thymidylate synthase, inhibiting the activity of thymidylate synthase, causing deoxynucleotide deficiency and DNA synthesis disorder. In addition, metabolites of 5-FU may also invade RNA and DNA in the form of pseudo metabolites, affect cellular functions, and cause cytotoxicity. 5-FU is an atypical cell cycle-specific drug that acts primarily on cells in other phases in addition to the S phase. The 5-fluorouracil has the side effects that: 1. the gastrointestinal reaction comprises anorexia, nausea, emesis, stomatitis, gastritis, and diarrhea, and severe cases comprise bloody diarrhea. 2. Bone marrow suppression: the white blood cells and the platelets are reduced, and when the white blood cells and the platelets are serious, the whole blood picture is reduced. 3. Local stimulation: the injection site can cause phlebitis. Arterial instillation can cause topical skin erythema, edema, ulceration, pigmentation. 4. The nervous system: a few may have cerebellar degeneration and ataxia. 5. Alopecia, dermatitis, nail bed darkening, and the like.

Cyclophosphamide, also called CTX, is an alkylating agent that was first synthesized in 1958. The traditional Chinese medicine composition is clinically used for malignant lymphoma, multiple myeloma, leukemia, breast cancer, ovarian cancer, cervical cancer, prostatic cancer, colon cancer, bronchial cancer, lung cancer and the like, and has certain curative effect. Cyclophosphamide has no antitumor activity in vitro, and is converted into aldehyde phosphoramide by microsome functional oxidase in liver after entering into body, and the aldehyde amide is unstable and decomposed into amide nitrogen mustard and acrolein in tumor cells, and the amide nitrogen mustard has cytotoxic effect on tumor cells. Cyclophosphamide is a bifunctional alkylating agent and a cell cycle nonspecific drug, can interfere with the functions of DNA and RNA, particularly has larger influence on the former, is cross-linked with the DNA to inhibit the DNA synthesis, and has the most obvious effect on the S phase. Cyclophosphamide has a number of side effects including: 1. gastrointestinal tract reaction: cyclophosphamide has significant gastrointestinal reactions such as anorexia, nausea and vomiting. 2. Blood system: cyclophosphamide can inhibit bone marrow and reduce leucocyte, and large dose of cyclophosphamide can cause thrombocytopenia and anemia. 3. Urinary system: patients sometimes have frequent, urgent and painful urination and hematuria after using cyclophosphamide, and hemorrhagic cystitis is easily caused when the cyclophosphamide is used at high concentration. 4. Increase in tumor incidence: leukemia, skin cancer, lymphoma, etc. can occur with long-term application of cyclophosphamide, and it has been reported that the incidence rate of bladder cancer in patients with long-term application of cyclophosphamide is 10 times higher than that in normal people. 5. And others: cyclophosphamide inhibits the development of ova, affects fertility, and can also cause side effects such as irregular menstruation, temporary alopecia, skin and nail pigmentation, etc.

In the innate immune pathway, microbial and viral DNA in infected mammalian cells can induce an endogenous, powerful immune response by stimulating interferon secretion. The immune response of the Endoplasmic Reticulum (ER) receptor protein (STING) to cytoplasmic DNA is an essential factor. Recent studies have shown that cyclic cGMP-AMP dinucleotide synthetase (cGAS) endogenously catalyzes cGAMP synthesis under activated conditions upon DNA binding. cGAMP is a cytosolic DNA sensor that acts as a second messenger to stimulate the induction of INF-beta by STING, mediating the activation of TBK1 and IRF-3, which in turn initiates transcription of the INF-beta gene. Recently, recombinant cGAS was reported to catalyze the cyclization of cGMP-AMP dinucleotide, gammp, under DNA binding conditions. The crystal structure of the complex where cGAS binds 18bp dsDNA has also been reported and studies of cGAMP in antiviral immunity have been demonstrated. cGAMP binds to STING, activates the transcription factor IRF3 and produces interferon beta, activates dendritic cells and T cells, and is able to activate immune response, so it is possible that cGAMP acts as a ligand of STING and can activate the immune system.

Disclosure of Invention

The invention aims to provide application of dinucleotide cGAMP in preventing and treating complications induced by antitumor chemotherapy and reducing toxic and side effects of chemotherapeutics.

The dinucleotide cGAMP disclosed by the invention is characterized by enhancing the curative effect of a chemotherapeutic drug on tumor treatment, treating complications induced by the chemotherapeutic drug and reducing the side effect of the chemotherapeutic drug.

The dinucleotide cGAMP of the invention includes cGAMP and its thio/seleno cGAMP derivatives.

In particular, the tumors include but are not limited to colorectal cancer cells, leukemia.

In particular, the tumors include but are not limited to colorectal cancer cells, leukemia; the antitumor drugs include but are not limited to 5-fluorouracil, cyclophosphamide; the side effect reduction comprises the reduction of intestinal mucosa injury and the increase of platelet number.

Reference herein to the dinucleotide cGAMP, unless otherwise specified, is intended to refer to 2 ', 5' -3 ', 5' -cGAMP Cyclic [ G (2 ', 5') pA (3 ', 5') p ] cGAMP (2 '-5') c [ G (2 ', 5') pA (3 ', 5') p ], CAS number 1441190-66-4.

Detailed description of the invention

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The first embodiment is as follows: cGAMP preparation

cGAMP (cyclic-GMP-AMP) is synthesized catalytically by cyclic cGMP-AMP dinucleotide synthetase (cGAS) under activating conditions after binding DNA according to literature procedures. The purity is more than 98%. (Pingwei Li, et al., Immunity,2013,39(6),1019-

Example two: establishment of tumor model of colorectal cancer of mouse and drug therapy

1. Establishment of tumor-bearing mouse model

C57BL/6 female mice, 6-8 weeks old, were purchased from Shanghai Spiker laboratory animals, Inc. The colorectal cancer MC38 cell is cultured and passaged according to a corresponding cell culture mode, and is used for establishing a tumor mouse model at a logarithmic phase. Cells were collected at the log phase of mouse colorectal cancer cells MC38 to a concentration of (1-5X 10)⁶) Per ml of cell suspension, C57BL/6 mice were injected with 0.2ml of MC38 cell suspension (cell number 2-10X 10)⁵One/one), the tumor grows to be about 3-4mm in diameter after about 7-9 days, and the tumorigenesis is successful.

After successful tumorigenesis, tumor-bearing mice were divided into 5 groups of 10 mice each at random. The five groups are respectively group A: negative control group (i.p. saline group), group B: group C, group d: group of positive plus low dose cGAMP (5-fluorouracil dose 10mg/kg, cGAMP dose 5mg/kg), group D: positive drug plus medium dose cGAMP group (5-fluorouracil dose 10mg/kg, cGAMP dose 10mg/kg), group E: positive drugs were added to the cGAMP group at a high dose (5-fluorouracil dose of 10mg/kg, cGAMP dose of 20 mg/kg). The positive drug and cGAMP were injected once daily into the tail vein.

After 20 days, mice were sacrificed and tumor bodies were weighed, and the effect of the tumor vaccine was observed, and the tumor inhibition rate was ═ 100% of [ 1-negative control group average tumor weight (B, C, D, E group is experimental group)/a group average tumor weight) ]. And (3) taking intestinal tract tissues of the mice, staining and slicing, comparing the intestinal tract mucosa injury conditions of different groups, and taking the intestinal tract mucosa staining of normal mice as a control.

2. Statistical analysis

Data are expressed in x ± s, treated with SPSS10.0 software, and the significance of tumor weight differences of each group was compared using one-way ANOVA (one-way ANOVA) test, with a significance level a of 0.05.

3. Results

After mice are inoculated with MC38 tumor cells of colorectal cancer subcutaneously, cGAMP combined with a positive drug 5-fluorouracil can obviously inhibit the growth of tumors, and the tumor weight average after 20 days of treatment is obviously lower than the single treatment effect of 5-fluorouracil without cGAMP (P <0.05, P <0.01), which shows that the cGAMP combined with 5-fluorouracil has the effect of enhancing the anti-tumor effect of 5-fluorouracil. Specific results are shown in table 1. Meanwhile, the damage of the positive drug 5-fluorouracil to the intestinal mucosa can be remarkably reduced by adding cGAMP, and the figure 1 shows.

TABLE 1 inhibitory Effect of cGAMP in combination with 5-fluorouracil on murine colorectal cancer cells MC38

(n＝10，mean±SD)

Note: p <0.05vs positive drug group; p <0.01vs positive drug group

Figure 1.cGAMP reduction of 5-fluorouracil injury to mouse intestinal mucosa results, from left to right: intestinal mucosa staining results of normal mice, mice with single cGAMP administration group, mice with positive drug 5-fluorouracil administration group, and mice with 5-fluorouracil and cGAMP administration group.

Example three: mouse leukemia model establishment and drug therapy

1. Establishment of leukemia model

DBA/2 female mice, 6-8 weeks old, were purchased from Shanghai Spiker laboratory animals, Inc. Leukemia L1210 cells are cultured and passaged according to corresponding cell culture modes, and are used for establishing a tumor mouse model at a logarithmic phase. Cells were collected at log phase of mouse L1210 cells to a concentration of (3X 10)⁷) Perml cell suspension, mice were injected intravenously with 0.1ml L1210 cell suspension (cell number 3X 10)⁶One/only).

Mice were randomly divided into 5 groups of 10 mice each. The five groups are respectively group A: negative control group (i.v. saline group), group B: group C, group d, group: group of positive drugs plus low dose cGAMP (cGAMP administration dose 5mg/kg), group D: positive drug plus medium dose cGAMP group (cGAMP dose 10mg/kg), group E: positive drugs were administered to a high-dose cGAMP group (cGAMP administration dose 20 mg/kg). The positive drug and cGAMP were injected intravenously once daily. The mean survival of the mice was observed. Blood was collected 10 days after the administration, and blood routine was measured while normal DBA/2 mice were taken as a control.

2. Statistical analysis

Data are expressed in x ± s, processed using SPSS10.0 software, and the significance of the differences in survival curves of the groups of mice was compared using a one-way ANOVA test, with a significance level a of 0.05.

3. Results

After mice are inoculated with L1210 leukemia cells by veins, the survival time of the mice can be obviously improved by the cGAMP combined cyclophosphamide, the survival time of the mice treated by the cGAMP combined cyclophosphamide for leukemia is obviously longer than that of a cyclophosphamide single treatment group (P <0.05, P <0.01), and the results show that: the cGAMP combines cyclophosphamide, which can obviously enhance the leukemia-resistant effect of cyclophosphamide. The specific results are shown in Table 2. And the cGAMP can inhibit the side effect of cyclophosphamide, and the number of platelets is closer to the normal number, and the result shows that: cGAMP can reduce partial side effect of cyclophosphamide and increase platelet number.

TABLE 2 mean survival of mice treated with cGAMP in combination with cyclophosphamide for leukemia

(n＝10，mean±SD)

Note: p <0.05vs cyclophosphamide positive control group; p <0.01vs cyclophosphamide positive control group.

TABLE 3 number of platelets in blood of leukemic mice receiving cGAMP in combination with cyclophosphamide therapy

(n＝10，mean±SD)

Note: p <0.05vs cyclophosphamide positive control group; p <0.01vs cyclophosphamide positive control group.

TABLE 4 number of erythrocytes in blood of leukemic mice receiving cGAMP in combination with cyclophosphamide treatment

(n＝10，mean±SD)

Note: p <0.05vs cyclophosphamide positive control group; p <0.01vs cyclophosphamide positive control group.

TABLE 5 number of neutrophils in blood of leukemic mice receiving cGAMP in combination with cyclophosphamide therapy

(n＝10，mean±SD)

Note: p <0.05vs cyclophosphamide positive control group; p <0.01vs cyclophosphamide positive control group.

Claims (1)

1. Use of c [ G (2 ', 5') pA (3 ', 5') p ] in combination with cyclophosphamide for the preparation of a medicament for the treatment of leukemia.