Ring dinucleotides cGAMP Application in the complication for preventing and treating antitumor chemical drug induction or the toxic and side effect for reducing chemotherapeutic inductionTechnical field
The invention belongs to biological medicine technology, immunotherapy of tumors field, and in particular to a kind of ring dinucleotides（cGAMP）And derivative is in therapeutic alliance tumour, the application reduced in antineoplastic toxicity.
Background technology
Tumour is one of major disease of a class serious harm human life and health, shows as cell hyperproliferation and disdifferentiation.WHO scholarly forecasts, the year two thousand twenty population in the world tumor invasion are up to 2 000 ten thousand people, and death toll is up to 1 200 ten thousand people, and tumour will become the first killer of this century mankind, constitute the threat of most serious to human survival.The M ＆ M of lung cancer, the knot/carcinoma of the rectum, cancer of the stomach, liver cancer etc. occupies the prostatitis of all kinds of malignant tumours.According to (2012 China's tumour registration annual reports that national tumour Register issues》Statistics, annual kainogenesis tumor cases about 3,120,000, average daily 8 550 people, the whole nation is per minute to have 6 people to be diagnosed as cancer.From the point of view of disease, lung cancer, cancer of the stomach, the knot/carcinoma of the rectum, liver cancer and the cancer of the esophagus occupy first five position of national malignant tumor morbidity.With the increase year by year of Cancer Mortality and the death rate, treating malignant tumor demand is increasing.
5 FU 5 fluorouracil, abbreviation 5-FU, 5 FU 5 fluorouracil are the fluoride of miazines, belong to antimetabolic antineoplastic, and thymidylate synthase, blocking deoxyribonucleotides nucleotides can be suppressed to be converted into thymidine core, interference DNA synthesis.Synthesis to RNA also has certain inhibitory action.It is clinically used for colon and rectum carcinoma, cancer of the stomach, breast cancer, oophoroma, chorioepithelioma, chorioadenoma, G. cephalantha, cutaneum carcinoma, liver cancer, carcinoma of urinary bladder etc..5-FU is first antimetabolite synthesized according to certain imagination and is clinically anti-miazines medicine most widely used at present have good efficacy to digestive system cancer and other solid tumors, occupy critical role in Internal Medicine-Oncology treatment.5 FU 5 fluorouracil need to have antitumor activity for the acid of 5- fluorodeoxyuridines through enzymatic conversion.5-FU suppresses the synthesis of DNA by suppressing thymidylate synthase.It is sour that the effect of this enzyme may be transferred to deoxyuridine-phosphoric acid synthesis thymidine one the one carbon unit of formyl tetrahydrofolic acid.Synthesis of the 5-FU to RNA also has certain inhibitory action.5-FU in vivo Jing after the biotransformation such as ribosylation and phosphorylated, just with cytotoxicity.5-FU can Jing different approaches generation F-dUMP and FUMP.The former can suppress the activity of this enzyme with the activated centre covalent bond of thymidylate synthetase, and acid heat to make deoxyribonucleoside, DNA dyssynthesises.Additionally, the metabolin of 5-FU can also pseudo- metabolite form enter in RNA and DNA disastrously, affect cell function, produce cytotoxicity.5-FU is a kind of atypical cell cycle specific medicine, and it also has effect to the cell of other phases in addition to mainly acting on the S phases.5 FU 5 fluorouracil side effect has：1. gastrointestinal reaction has anorexia, Nausea and vomiting, stomatitis, gastritis, diarrhoea, and severe patient has bloody diarrhea.2. bone marrow suppression：Leucocyte and blood platelet decline, and can have whole blood as declining when serious.3. local excitation：Injection site can cause phlebitis.Artery instils and can cause local skin erythema, oedema, ulceration, pigmentation.4. nervous system：Minority can have cerebellar degeneration, incoordination.5. alopecia, dermatitis, nail matrix blackening etc..
Endoxan, is called CTX, is a kind of alkylating agent, and 1958 artificial synthesized first.Malignant lymphoma, Huppert's disease, leukaemia, breast cancer, oophoroma, cervical carcinoma, prostate cancer, colon cancer, bronchiolar carcinoma, lung cancer etc. are clinically used for, have certain curative effect.Endoxan is in vitro without antitumor activity, aldophosphamide is changed into into the rear first Jing particulates body function oxidizing ferment in liver in vivo, and aldehyde acid amides is unstable, acid amides mustargen and methacrylaldehyde are resolved in tumour cell, acid amides mustargen has CDCC to tumour cell.Endoxan is bifunctional alkylating agents and cell cycle nonspecific agent (CCNSA), may interfere with DNA and RNA functions, especially bigger with the impact to the former, and it occurs cross link with DNA, suppresses DNA synthesis, acts on most obvious to the S phases.Endoxan has many side effects, including：1st, gastrointestinal reaction：Endoxan has obvious gastrointestinal reaction, such as loses the appetite, n and V etc..2nd, hematological system：Marrow can be suppressed using endoxan, make Neuroleptic Leukocytopenia, heavy dose of use cause decrease of platelet, anaemia.3rd, urinary system：Patient using urine urgency-frequency odynuria sometimes after endoxan, it may also occur that blood urine, when high concentration is used, easily causes hemorrhagic cystitis.4th, increase tumor incidence：Prolonged application endoxan can occur leukaemia, skin cancer, lymthoma etc., have been reported that the incidence of disease of patient's carcinoma of urinary bladder of prolonged application endoxan is higher than 10 times of normal person.5th, other：Endoxan can suppress egg cell development, affect fertility, can also result in the side effects such as irregular menstruation, temporary alopecia, skin, nail pigmentation.
In innate immunity path, in the mammalian cell of infection, microorganism and viral DNA can be by stimulating the strong immune response in interferon secretion induction Inner sources.Endoplasmic reticulum（ER）Receptor protein（STING）It is required factor to the immune response of cytoplasmic DNA.It has recently been demonstrated that cyclisation cGMP-AMP dinucleotides synzyme（cGAS）Under activation condition after with reference to DNA, the synthesis of cGAMP is endogenously catalyzed.CGAMP is a kind of cytoplasmic DNA sensor, and it stimulates the sensing of INF- β as second messenger by STING, mediates the activation of TBK1 and IRF-3, and then starts the transcription of INF- β genes.Report recently, recombinate cGAS catalytic cyclization cGMP-AMP dinucleotides GAMP under DNA conjugation conditions.CGAS has also been reported with reference to the crystal structure of the compound of 18bp dsDNA, and researchs of the cGAMP in terms of antiviral immunity has been found to.CGAMP combines STING, makes transcription factor IRF3 activate and produce IFN-β, dendritic cell activated and T cell, can activate immune response, it is thus possible to parts of the cGAMP as STING, can be with activating immune system.
The content of the invention
It is an object of the invention to provide a kind of applications of dinucleotides cGAMP in the complication and reduction chemical drug toxic and side effect of preventing and treating anti-tumor chemotherapeutic induction.
The dinucleotides cGAMP of the present invention, it is characterised in that can strengthen chemotherapeutic drug therapy tumor efficiency, treats because of the complication of chemotherapeutic induction, reduces chemotherapeutics side effect.
The dinucleotides cGAMP of invention, including cGAMP and its thio/seleno cGAMP derivatives.
The another further aspect of invention is related to heretofore described cGAMP and is preparing the toxic and side effect or complication of antitumor chemical drug induction. and specifically, the tumour includes but is not limited to colorectal cancer cell, leukaemia.
The another further aspect of invention is related to heretofore described cGAMP and is preparing the toxic and side effect or complication of antitumor chemical drug induction. and specifically, the tumour includes but is not limited to colorectal cancer cell, leukaemia；The antineoplastic includes but is not limited to 5 FU 5 fluorouracil, endoxan；Described reduction side effect includes that reducing intestinal mucosa damages, lifts platelet counts.
Dinucleotides cGAMP is mentioned above, specified otherwise is not added with such as, refer both 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], No. CAS is 1441190-66-4, and molecular formula is C20H22N10O13P2 .2NH4。
Specific implementation method
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Embodiment one：It is prepared by cGAMP
cGAMP （Cyclisation-GMP-AMP）Under by activation condition of the literature method after with reference to DNA, by cyclisation cGMP-AMP dinucleotides synzyme（cGAS）Catalyze and synthesize.Purity is more than 98%.（Pingwei Li, et al., Immunity, 2013,39(6), 1019-1031.）
Embodiment two：Mouse colorectal cancer tumor model is set up and drug therapy
1. The foundation of lotus knurl mouse model
C57BL/6 female mices, 6-8 week old, are purchased from Shanghai Slac Experimental Animal Co., Ltd..The cell culture of colorectal cancer MC38 to pass on reference to the culture of corresponding cell culture mode with pass on, and logarithm period be used for tumor mouse model foundation.Cell is collected in mouse colorectal cancer cell MC38 logarithmic phases, make the cell suspension that concentration is (1-5 × 10e6)/per milliliter of ml, inject 0.2 ml MC38 cell suspensions in C57BL/6 mouse right fores oxter (cell number is 2-10 × 10e5 /), 7-9 days or so tumour length is to diameter about 3-4mm, tumorigenesis success.
After tumorigenesis success, mice with tumor is divided into 5 groups at random, 10 per group.Five groups is A groups respectively：Negative control group（Intraperitoneal injection of saline group）, B groups：Positive drug group（5 FU 5 fluorouracil, 10 mg/kg of dosage）, C groups：Positive drug adds low dosage cGAMP groups（10 mg/kg of 5 FU 5 fluorouracil dosage, 5 mg/kg of cGAMP dosages）, D groups：Positive drug adds middle dosage cGAMP group（10 mg/kg of 5 FU 5 fluorouracil dosage, 10 mg/kg of cGAMP dosages）, E groups：Positive drug increases dosage cGAMP groups（10 mg/kg of 5 FU 5 fluorouracil dosage, 20 mg/kg of cGAMP dosages）.Positive drug and the daily tail vein injections of cGAMP are once.
After 20 days, put to death mouse and claim tumor weight, the effect of observation tumor vaccine, tumour inhibiting rate=[the average knurl weight of 1- negative control groups (B, C, D, E group is the experimental group)/average knurl weight of A groups)] × 100%.Mouse intestinal tissue, dyeing are taken, and makes section, relatively different groups of intestinal mucosa degree of impairments, while the intestinal mucosa dyeing of normal mouse is taken as control.
2. statistical analysis
Data are represented with x ± s, are processed using SPSS10.0 softwares, using one-way analysis of variance（one-way ANOVA）The conspicuousness of each group knurl weight difference, significance a=0.05 are compared in inspection.
3.As a result
After mouse hypodermic inoculation colorectal cancer MC38 tumour cell, cGAMP joint positive drug 5 FU 5 fluorouracils can substantially suppress tumour growth, and the knurl weight after treating 20 days is substantially less than the 5 FU 5 fluorouracil monotherapy effect for not adding cGAMP（P<0.05, P<0.01）, show cGAMP joint 5 FU 5 fluorouracils, with enhancing 5 FU 5 fluorouracil antitumor curative effect.Concrete outcome is shown in Table 1.Meanwhile, add cGAMP significantly reduce damage of the positive drug 5 FU 5 fluorouracil to intestinal mucosa, see Fig. 1.
Table1 cGAMPJoint5-Fluorouracil is to mouse colorectal cancer cellMC38Inhibition
（N=10, mean ± SD）
GroupAverage knurl weight（g）Average tumour inhibiting rate（%）
Negative control group2.434±0.179 （g）
Positive drug group（5 FU 5 fluorouracil）1.263±0.231 （g）48.1
Positive drug adds low dosage cGAMP groups1.048±0.163 （g）56.9
Positive drug adds middle dosage cGAMP group0.727±0.214 （g）*70.1
Positive drug adds middle dosage cGAMP group0.581±0.244 （g）**76.1
Note：*P<0.05 vs positive drug groups；**P<0.01 vs positive drug groups
Fig. 1. cGAMP reduces 5 FU 5 fluorouracil to mouse intestinal mucosa injury result, is respectively from left to right：Normal mouse enteron aisle, cGAMP are administered alone a group mouse intestinal mucosa dyeing result, positive drug 5 FU 5 fluorouracil administration group mouse intestinal mucosa dyeing result, 5 FU 5 fluorouracil joint cGAMP administration group mouse intestinal mucosa dyeing results.
Embodiment three：Mouse leukemia model is set up and drug therapy
1.The foundation of Leukemia Model
DBA/2 female mices, 6-8 week old, are purchased from Shanghai Slac Experimental Animal Co., Ltd..Ehrlich tumor cell culture to pass on reference to the culture of corresponding cell culture mode with pass on, and logarithm period be used for tumor mouse model foundation.Cell is collected in the mouse L1210 cell log phases, the cell suspension that concentration is (3 × 10e7)/per milliliter of ml, 0.1 ml L1210 cell suspensions of mouse mainline is made (cell number is 3 × 10e6 /).
Mouse is divided into 5 groups at random, 10 per group.Five groups is A groups respectively：Negative control group（Intravenous injection physiological saline group）, B groups：Positive drug group（Endoxan, 10 mg/kg of dosage）, C groups：Positive drug adds low dosage cGAMP groups（5 mg/kg of cGAMP dosages）, D groups：Positive drug adds middle dosage cGAMP group（10 mg/kg of cGAMP dosages）, E groups：Positive drug increases dosage cGAMP groups（20 mg/kg of cGAMP dosages）.Positive drug and cGAMP intravenous injection daily is once.Observation mouse mean survival time (MST).Take a blood sample after being administered 10 days, determine routine blood test, while normal DBA/2 mouse are taken as control.
2. statistical analysis
Data are represented with x ± s, are processed using SPSS10.0 softwares, using one-way analysis of variance（one-way ANOVA）The conspicuousness of each group mouse survival curve difference, significance a=0.05 are compared in inspection.
3.As a result
After mouse vein inoculation L1210 leukaemia, cGAMP joint endoxan can significantly improve survival time of mice, and cGAMP joint treated with cyclophosphamide pulse leukemia mouse life cycles are considerably longer than the life cycle of endoxan monotherapy group（P<0.05, P<0.01）, as a result show：CGAMP combines endoxan, can significantly increase endoxan anti-leukocythemia curative effect.Concrete outcome is shown in Table 2.And cGAMP can suppress the side effect of endoxan, platelet counts as a result to show closer to normal number：CGAMP can reduce the part side effect of endoxan, improve platelet counts.
Table2 cGAMPJoint treated with cyclophosphamide pulse leukemia mouse mean survival time (MST)
（N=10, mean ± SD）
GroupMean survival time (MST)（My god）
Negative control group9±1.2 （My god）
Positive drug group（Endoxan）23.2±2.5 （My god）
Positive drug adds low dosage cGAMP groups24.1±1.9 （My god）
Positive drug adds middle dosage cGAMP group27.8±1.2 （My god）*
Positive drug increases dosage cGAMP groups30.6±2.1 （My god）**
Note：*P<0.05 vs endoxan positive controls；**P<0.01 vs endoxan positive controls.
Table3ReceivecGAMPPlatelet counts in the blood of the leukemia mouse of joint treated with cyclophosphamide pulse
（N=10, mean ± SD）
Group Platelet count（10e10/L）
Normal mouse126±27 -
Positive drug group（Endoxan） 27±17
Positive drug adds low dosage cGAMP groups59±13 *
Positive drug adds middle dosage cGAMP group77±24 **
Positive drug increases dosage cGAMP groups94±36 **
Note：*P<0.05 vs endoxan positive controls；**P<0.01 vs endoxan positive controls.
Table4ReceivecGAMPErythrocyte number in the blood of the leukemia mouse of joint treated with cyclophosphamide pulse
（N=10, mean ± SD）
GroupRed blood cell number（10e12/L）
Normal mouse8.5±1.4 -
Positive drug group（Endoxan） 4.2±1.1
Positive drug adds low dosage cGAMP groups4.7±1.3
Positive drug adds middle dosage cGAMP group5.9±0.8 *
Positive drug increases dosage cGAMP groups7.8±1.1 **
Note：*P<0.05 vs endoxan positive controls；**P<0.01 vs endoxan positive controls.
Table5ReceivecGAMPNeutrophil leucocyte quantity in the blood of the leukemia mouse of joint treated with cyclophosphamide pulse
（N=10, mean ± SD）
GroupNeutrophil leucocyte number（10e9/L）
Normal mouse3.9±1.5 -
Positive drug group（Endoxan）1.2±0.3
Positive drug adds low dosage cGAMP groups1.4±0.2
Positive drug adds middle dosage cGAMP group1.9±0.4 *
Positive drug increases dosage cGAMP groups3.2±0.5 **
Note：*P<0.05 vs endoxan positive controls；**P<0.01 vs endoxan positive controls.