CN106928298B

CN106928298B - Structural composition of cyclic dinucleotide cGAMP derivative, preparation method and application of cyclic dinucleotide cGAMP derivative in tumor resistance

Info

Publication number: CN106928298B
Application number: CN201710147711.5A
Authority: CN
Inventors: 张跃茹; 向道凤; 谭瀛轩; 谭相石
Original assignee: Hangzhou Xing'ao Biological Technology Co ltd
Current assignee: HANGZHOU XING'AO BIOLOGICAL TECHNOLOGY Co.,Ltd.
Priority date: 2017-03-13
Filing date: 2017-03-13
Publication date: 2021-10-22
Anticipated expiration: 2037-03-13
Also published as: CN106928298A

Abstract

The invention relates to the technical field of biological medicines, and prepares an innovative derivative of serial cyclic dinucleotide cGAMP, wherein the derivative of the serial cGAMP has a remarkable anti-tumor effect and has important potential application in anti-tumor and anti-tumor drug preparation.

Description

Structural composition of cyclic dinucleotide cGAMP derivative, preparation method and application of cyclic dinucleotide cGAMP derivative in tumor resistance

Technical Field

The invention relates to the technical field of biological medicines, in particular to an innovative derivative of serial cyclic dinucleotide cGAMP, which has obvious anti-tumor effect and important application in anti-tumor and anti-tumor drug preparation.

Background

Malignant tumors are common frequently encountered diseases seriously threatening human health, and become an important cause of human death. Currently, the main treatment means for tumors include drug therapy, surgical treatment and radiation therapy. Although novel antitumor drugs represented by molecular targeted drugs are emerging continuously, the conventional cytotoxic antitumor drugs still have a dominant position in the drug therapy of tumors in a considerable period of time.

The traditional cytotoxic antitumor drug directly inhibits the proliferation of tumor cells or induces the apoptosis of tumor cells mainly by influencing the structures and functions of nucleic acids and proteins of the tumor cells. Despite the efforts made in the drug treatment of tumors to study new chemotherapeutic drugs and to improve chemotherapeutic regimens, the therapeutic effect is still not ideal. The traditional cytotoxic antitumor drug lacks an ideal selection effect on tumor cells and normal cells, and has damage to normal tissues of a human body while killing malignant tumor cells, so that serious systemic adverse reactions are caused, wherein the common adverse reactions are bone marrow suppression, digestive tract reaction and alopecia. Many patients are unable to adhere to, and even forego, chemotherapy, and it is clinically difficult to expect the desired effect by increasing the dose of the drug. These factors severely affect the efficacy of chemotherapy.

Recent studies have shown that cyclic dinucleotide synthetase (cGAS) catalyzes cGAMP synthesis under activated conditions after binding to DNA, further mediating IRF-3 activation through STING, and further promoting IFN- β synthesis. Exogenous administration of recombinant cGAS promotes the generation of cyclic dinucleotide cGAMP under the DNA binding condition, and plays roles in resisting virus and enhancing immunity. In vitro synthesized cyclic dinucleotide cGAMP has also been reported in patents for antitumor applications. cGAMP, a cyclic-di-phosphorothioate, has better antitumor activity than cGAMP.

The invention prepares the innovative derivatives of the serial cyclic dinucleotide 2 '3' -cGAMP, and the serial derivatives have better stability and affinity with target protein STING than the cGAMP, have better anti-tumor effect and have important potential application in anti-tumor and anti-tumor drug preparation.

Disclosure of Invention

The technical scheme of the invention provides the structural composition and the preparation method of serial derivatives of cyclic dinucleotide cGAMP and the application of the serial derivatives in antitumor and antitumor drug preparation.

The structural composition of cyclic dinucleotide cGAMP-XYZ derivatives mainly refers to cGAMP derivatives prepared by modifying and transforming 2 '3' -cGAMP. The following categories are classified:

(1)2 '3' -cGAMP-X, derivatives of cGAMP having a substituent modified at the adenine moiety (2-R1/8-R2, R1/R2 ═ amino, halogen, methoxy, etc.)

(2)2 '3' -cGAMP-Y, derivatives of cGAMP with a modified guanine moiety (8-R3, R3 ═ amino, halogen, methoxy, etc.)

(3)2 '3' -cGAMP-XY, derivatives of cGAMP with substituents on both the adenine and guanine moieties (2-R1/8-R2, R1/R2 ═ amino, halogen, methoxy, etc.; 8-R3, R3 ═ amino, halogen, methoxy, etc.)

(4)2 '3' -cGAMP-XZ, cGAMP having substituent modifications on the adenine moiety, while the phosphate of the cyclic phospholipidic linkage moiety has sulfur-or selenium-substituted derivatives.

(5)2 '3' -cGAMP-YZ, cGAMP has substituent modification at guanine part, and the phosphate radical of cyclic phosphate ester bond part has sulfur or selenium substituted derivative.

(6)2 '3' -cGAMP-XYZ, cGAMP has substituent modifications on both adenine and guanine moieties, while the phosphate of the cyclic phosphate linkage moiety has a sulfur-or selenium-substituted derivative.

A preparation method of 2 '3' -cGAMP-XYZ derivatives comprises the following steps:

(1) adenosine triphosphate ATP and guanosine triphosphate GTP substituent derivatives, catalyzed by cyclic dinucleotide synthetase (cGAS) activated upon binding duplex DNA, to prepare 2 '3' -cGAM-XY derivatives (including: 2 '3' -cGAMP-X, 2 '3' -cGAMP-Y, 2 '3' -cGAMP-XY);

(2) derivatives of the substituents of α -phosphothio-substituted (selenium-substituted) adenosine triphosphate ATP and guanosine triphosphate GTP) are catalyzed by cyclic dinucleotide synthetase (cGAS) activated after binding to duplex DNA to prepare α -phosphothio-substituted (selenium) -2 '3' -cGAMP derivatives (including: 2 '3' -cGAMP-XZ, 2 '3' -cGAMP-YZ, 2 '3' -cGAMP-XYZ);

the 2 '3' -cGAMP derivative is used in antitumor, and is one or several of medicine, reagent and other types.

In the present invention, the tumor includes, but is not limited to, colon cancer, lung cancer, ovarian cancer, melanoma, gastric cancer, etc.

The antitumor drug prepared from the 2 '3' -cGAMP derivative can be prepared into various dosage forms according to conventional pharmacy, including tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections and the like.

The 2 '3' -cGAMP derivative antitumor agent can be used for preventing or treating tumor-related diseases by adopting one or more administration routes of oral administration or injection (comprising one or more of intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection and the like).

The 2 '3' -cGAMP derivative antitumor agent provided by the invention has good application prospect in the drug treatment of tumors.

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are given to better illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1: cyclic dinucleotide cGAMP derivative composition and preparation method

(1) Various ATP and GTP derivatives are purchased from Sigma, usa. Derivatives of ATP and GTP are as follows:

(a) ATP-X, ATP substituent-modified derivatives (2-R1/8-R2, R1/R2 ═ amino, halogen, methoxy, and the like)

(b) GTP-Y, GTP substituent-modified derivatives (8-R3, R3 ═ amino, halogen, methoxy, etc.)

(2) Adenosine 5 '- α -thio (seleno) phosphate and guanosine 5' - α -thio (seleno) phosphate were prepared according to literature methods. (HUANGZHen et al, Science China, Chemistry,2012,55(1),80-89, BoyleN.A., et al, Nucleic Acids and Nucleic Acids,2005,24, 1651-.

(3) Preparation of 2 '3' -cGAMP derivatives and 2 '3' -cGAMP derivatives

Referring to the preparation method of 2 '3' -cGAMP (PingweiLi, et al., Immunity,2013,39(6),1019-1031.), the synthesis of 2 '3' -cGAMP-XY derivatives (including: 2 '3' -cGAMP-X, 2 '3' -cGAMP-Y, 2 '3' -cGAMP-XY) is catalyzed by cyclic dinucleotide synthetase (cGAS) under the activation condition after binding DNA instead of ATP and GTP.

(4) Preparation of 2 '3' -cGAMP-XYZ derivatives of phosphorothioic acid (seleno), the adenosine derivatives 5 '-a-thio (seleno) phosphate and guanosine derivatives 5' -a-thio (seleno) phosphate were prepared according to literature procedures, with reference to the preparation of 2 '3' -cGAMP. (HUANGZHen et al, Science China, Chemistry,2012,55(1),80-89., Boyle N.A., et al, Nucleic Acids and Nucleic Acids,2005,24,1651-1664.) Synthesis of 2 '3' -cGAMP derivatives of phosphorothioated (seleno) phosphate (including 2 '3' -cGAMP-XZ, 2 '3' -cGAMP-YZ, 2 '3' -cGAMP-XYZ) under activation conditions in combination with DNA binding, catalyzed by cyclodioic acid synthetase (cGAS), has a purity of 98% or more.

Example 2: cyclic dinucleotide cGAMP derivatives for antitumor animal experiments

The tumor-bearing mouse model is adopted to detect the inhibition effect of the 2 '3' -cGAMP derivative on the growth of subcutaneous transplanted tumors of animals and the toxic effect on the animals.

The 2 '3' -cGAMP derivatives used for antitumor experiments were selected from the following five classes:

(1) cGAMP-X1(2 '3' -amino-substituted derivative at adenine 2 position in cGAMP)

(2) cGAMP-Y1 (guanine 8-amino substituted derivative of 2 '3' -cGAMP)

(3) cGAMP-X1Y1(2 '3' -amino-substituted derivatives of cGAMP at the adenine 2-position and guanine 8-position)

(4) cGAMP-X1Z1 (a sulfur phosphate substituted derivative of cGAMP-X1)

(5) cGAMP-X1Y1Z1 (a sulfur phosphate substituted derivative of cGAMP-X1Y 1)

BALB/C normal mice, C57/BL6 normal mice, male, 16-18g in weight, 6-8 weeks old, SPF grade, purchased from shanghai slaike laboratory animals llc [ laboratory animal quality certification no: SCXK (Shanghai) 2007 + 0005 ].

Feeding conditions

All mice were left to eat and drink water freely, and were bred at room temperature (23 + -2) deg.C in the laboratory animal center of the university of civil liberation military and military medical sciences of China. The feed and water are sterilized by high pressure, and the whole experimental feeding process is SPF grade.

Dose setting

I.v. mice, 1 dose group was set: 10mg/kg

Test control

Negative control: physiological saline solution

Positive control: cGAMP at a dose of 10mg/kg

Method of administration

The administration route is as follows: tail vein injection administration

Administration volume: 100 microliter/piece

The administration times are as follows: 1 time per day for 21 days

Number of animals per group: 10 pieces of

Tumor cell strain

Mouse colorectal cancer cell line CT26, mouse lung cancer Lewis tumor line LL/2, human ovarian cancer cell line SK-OV-3, human melanoma cell line A375 and human gastric cancer cell line MNK-45 are purchased from cell banks of Chinese academy of sciences.

The main steps of the test

1. Establishment and intervention of tumor model mouse

The cells are cultured, passaged, collected in a cell log phase, made into cell suspension with the concentration of (1.0 multiplied by 107) per milliliter, and injected into the armpit of the right forelimb of the mouse with 0.2ml of cell suspension (the number of the cells is 2.0 multiplied by 106/mouse), the tumor grows to the diameter of about 5mm after about 10 days, the tumorigenesis is successful, and the cells are randomly divided into 5 groups. Respectively is A: negative control group (i.v. saline group); b: cGAMP group (intravenous cGAMP)10 mg/kg; C. d, E, F, G: group of 2 '3' -cGAMP derivatives (intravenous injection of 2 '3' -cGAMP derivatives) 10 mg/kg. The administration is 1 time per day for 21 days. After 21 days, mice were sacrificed and tumor body weight was weighed, and tumor inhibition rate ═ 1-mean tumor weight in experimental group (B, C, D, E, F, G group)/mean tumor weight in group a) ] x 100%.

Preparing subcutaneous transplantation tumor models respectively: mouse colorectal cancer cell line CT26, mouse lung cancer Lewis tumor line LL/2, human ovarian cancer cell line SK-OV-3, human melanoma cell line A375 and human gastric cancer cell line MNK-45, and observing the anti-tumor effect of the cGAMP derivative.

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.

Results

After mice are inoculated with tumor cells subcutaneously to prepare a successful subcutaneous transplantation tumor model, the 2 '3' -cGAMP-XYZ derivative can obviously inhibit the tumor growth, the tumor weight average after 21 days of administration is obviously lower than that of a negative control group (P <0.05, P <0.01), and 2 '3' -cGAMP-XYZ is better than that of cGAMP taken alone, which shows that 2 '3' -cGAMP-XYZ has better anti-tumor effect. Specific results table 1-table 5:

TABLE 1 Effect of cGAMP-XYZ on murine colorectal cancer cells CT26 subcutaneous transplantation tumor of BalB/C (mean. + -. SD, n 10)

Note: p <0.05vs negative control group; p <0.01vs negative control group.

TABLE 2 Effect of cGAMP-XYZ on C57 mouse Lung cancer Lewis tumor strain LL-2 subcutaneous transplantation tumor (n 10, mean + -SD)

Note: p <0.05vs negative control group; p <0.01vs negative control group.

TABLE 3 Effect of cGAMP-XYZ on human melanoma cell line A375 murine subcutaneous transplantation tumor (mean. + -. SD ═ 10)

Note: p <0.05vs negative control group; p <0.01vs negative control group.

TABLE 4 Effect of cGAMP-XYZ on murine subcutaneous transplantable tumor of human gastric carcinoma cell line MNK-45 (mean + -SD, n 10)

Note: p <0.05vs negative control group; p <0.01vs negative control group.

TABLE 5 Effect of cGAMP-XYZ on human ovarian cancer cell SK-OV-3 murine subcutaneous transplantation tumor (mean + -SD, n 10)

Note: p <0.05vs negative control group; p <0.01vs negative control group.

Example 3 acute toxicity study of cGAMP derivatives

Experimental Material

20 ICR mice (purchased from Shanghaisleke laboratory animals, Limited liability company [ laboratory animal quality certification number: SCXK (Shanghai) 2007-0005]), each half of male and female, the weight of the mice is 18-22 g, and the mice are fed with pellet feed and can freely eat and drink water.

cGAMP derivatives were prepared from example 1 and formulated into a solution at a concentration of 200mg/mL with physiological saline.

Experimental methods

ICR mice were injected with 2g/kg of cGAMP derivative sustained-release drug by single tail vein injection according to body weight, and the mice were observed for toxicity and death within 14 days after administration. As a result, it was found that the mice were normally active after a single tail vein injection administration. Within 14 days after administration, the mice did not die, and on day 15, all mice were sacrificed, dissected, and examined by naked eyes for each organ, and no obvious lesion was observed.

Results of the experiment

The result of the acute toxicity test shows that the maximum tolerance MTD of intravenous injection administration is not less than 2g/Kg, which indicates that the acute toxicity of the cGAMP derivative medicine is low.

Claims

The molecular structure composition of 2 '3' -cGAMP derivatives:

the 2 '3' -cGAMP derivatives prepared by modifying cyclic dinucleotide 2 '3' -cGAMP are as follows:

2 '3' -cGAMP-XYZ, cGAMP has amino substitution modification at both adenine 2-position and guanine 8-position, and the phosphate radical of cyclic phosphate ester bond part has sulfur substituted derivative.
2. An antitumor agent prepared by using the 2 '3' -cGAMP derivative of claim 1, wherein: the 2 '3' -cGAMP derivative preparation prepared by unit preparations containing the 2 '3' -cGAMP derivatives with different specifications and pharmaceutically acceptable carriers is one or more of tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections, and adopts one or more administration routes of oral administration or injection to prevent or treat tumors and directly related diseases thereof.
3. Use of the 2 '3' -cGAMP derivative of claim 1 for the manufacture of a medicament for the prevention and treatment of tumors and diseases directly related thereto.
4. Use of the 2 '3' -cGAMP derivative of claim 1 for the preparation of a combination with an anti-neoplastic agent.
5. Use of the 2 '3' -cGAMP derivative of claim 1 for the preparation of a medicament for combination with an anti-tumor monoclonal antibody.