CN101712710A - LMP1targeted oligonucleotide and application thereof serving as radiotherapeutic sensitizer - Google Patents
LMP1targeted oligonucleotide and application thereof serving as radiotherapeutic sensitizer Download PDFInfo
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Abstract
The invention relates to an EBV virus LMP1 targeted oligonucleotide and also relates to application of the oligonucleotide in treatment and radiotherapeutic sensitization of nasopharyngeal darcinoma. The oligonucleotide has the following characteristics that: (1) the oligonucleotide is complementary with an EBV LMP1 gene through the oligonucleotide binding sequence; (2) the oligonucleotide consists of 11-70 ribonucleotides, and the ribonucleotides are connected by organic phosphate bonds from the 5' direction to the 3' direction; (3) the 2' site of ribose has a methoxy-substituted radical; and (4) the 3' end is connected with a butanol radical. The modified oligonucleotides have relatively high enzymolysis resistance and biological activity, and can serve as an effective medicine for treating or assisting to treat nasopharyngeal darcinoma or EBV relevant diseases.
Description
Technical field
The present invention relates to the oligonucleotide of a kind of target EBV virus LMP1.
The invention still further relates to the application of this oligonucleotide in nasopharyngeal carcinoma treatment and radio therapy sensitization.
Background of invention
(nasopharyngeal carcinoma is a kind of epithelial cell malignant tumour with race and regional distribution difference NPC) to nasopharyngeal carcinoma, and sickness rate is higher more than 100 times than white race crowd in the southern china crowd, is 25-30/10 ten thousand.NPC is one of China and the modal malignant tumour of south east asia, all there is morbidity in each province and city in China, the coastal south is apparently higher than the north and interior ground, betide any age, the male sex is higher than the women, the Childhood rare, poly-liter of 20-40 year age group sickness rate, 40-60 year is the peak period, descends gradually later in 70 years old.NPC is the malignant tumour that the pharynx nasalis mucous epithelium takes place.Clinically, radiotherapy is first-selected therapeutic modality, but often causes the treatment failure because of the radiotherapy opposing.
EBV (Epstein-Barr virus) is a kind of gamma herpes viruses, the about 170kb of viral DNA total length.It is very extensive that epidemiological study shows that EBV infects, and adult infection rate can reach 98%; In developing country, the infection of EBV betides the childhood (before 5 years old) usually, and in developed country, the infection pilosity of EBV is born in pubescence.EBV infects and exists with two kinds of forms: latent infection and cracking are duplicated.Cracking is duplicated and is made Apoptosis of Host Cells, and latent infection makes virus and host cell long-term co-existence.In most of the cases, exist with the latent infection state behind the EBV infection host.In nasopharyngeal carcinoma, main EBNA1, LMP1, LMP2, the BamH I A of expressing then expresses all antigens in the Hodgkin disease in the lymphoma after transplanting.In latent state, viral DNA is present in the cell with a kind of form of episome mostly, when division takes place in host cell, carry out duplicating of DNA synchronously with the karyomit(e) of cell, and with the chromosomal separation of host cell in uniformly distributing to the two filial generation cell, thereby unlikely generation EBV's loses in daughter cell.
The Epstein-Barr virus latent infection is main with nasopharyngeal carcinoma, Burkitt lymphoma, many tumor developments such as relevant bone-marrow-derived lymphocyte knurl, t cell lymphoma, Hodgkin are closely related with HIV infection or other immunosuppressant diseases.The nearest Epstein-Barr virus of discovering also has certain relation with other tumours, for example, and lung cancer, cancer of the stomach, mammary cancer, cholangiocarcinoma, leiomyoma, colorectal carcinoma, prostate cancer etc.
LMP1 (latent membrane protein 1) is the tumorigenesis albumen with tumor gene function of having been proved conclusively at present of EBV coding, plays an important role in the morbidity of EBV related neoplasms.The gene of coding LMP1 is BNLF1, and it is transcribed by ED-L1 and L1-TR and activates sub control.The transmembrane protein that LMP1 is made up of 386 amino-acid residues, the about 62kD of its molecular weight comprises three kinds of different structural domains: 1) hydrophilic ammonia cardinal extremity cytoplasmic domain; 2) 6 hydrophobicitys are striden the film district, and these two zones are relevant with location and the polymerization of LMP1 albumen on film; 3) hydrophilic ammonia cardinal extremity carboxyl district.C-terminal has three structural domains and LMP1 function closely related.Be positioned at the CTAR1 of 194-231 amino acids residue,, regulate the activity of AP-1 activity and about 20-30%NF-κ B by acting on mutually with TRAFs.The CTAR2 that is positioned at 351-386 amino acids residue directly acts on by the mixture with TRADD and TRAF2 formation, induces the activation of 70-80%NF-κ B.And the CTAR3 that the 232-351 amino acids residue between the above two is formed, may be relevant with the JAK/STATs transcriptional activation.
LMP1 has multiple important biological function, studies confirm that LMP1 mainly mediates the cross-talk between NF-κ B, AP-1 and STAT three bars conduction paths and the signal transduction pathway, cause cell cycle disorder, apoptosis retardance, uncontrolled cellular proliferation and radiotherapy opposing.Morbidity and the EBV of NPC are closely related, and the LMP1 positive rate is up to 72% in patient NPC.In the clinical study of LMP1 and NPC relation, find to alleviate fully behind the LMP1 male NPC radical radiation therapy (complete remission, CR) rate is 69.0%, negative patient is 100.0%, two group relatively significant difference (P＜0.05).LMP1 positive cases distant metastasis rate is 31.0% (9/29), and negative patient is 0.0%.Can think that LMP1 not only has vital role in the NPC morbidity, and be the important factor of judging patient's radiotherapy effect and prognosis.Therefore, seek the radio therapy sensitization strategy of target LMP1, improve radiotherapy effect and will have the important clinical meaning.
The applicant specifically discloses the DNAzyme of not modified target LMP1 in Chinese patent (application number 200610032257.0).The application is the continuation research on this Chinese patent application basis.The oligonucleotide structure of target LMP1 after the preferred modification of research.
Summary of the invention:
The oligonucleotide that the purpose of this invention is to provide the modified target LMP1 of a class, oligonucleotide after such is modified has higher relatively resistance to enzymolysis ability and biological activity, and this oligonucleotide can be as the medicine of effective treatment or assisting therapy nasopharyngeal carcinoma or EBV relative disease.
Oligonucleotide provided by the invention has following feature:
(1) by its binding sequence and EBV LMP1 gene complementation;
(2) form by 11～70 Nucleotide, from 5 ' to 3 ' direction, connect by the phosphoric acid ester bond;
(3) on ribose 2 ' position, have the methoxyl group substituted radical;
(4) at 3 ' the terminal butanols group that connects.
Oligonucleotide of the present invention is a DNAzyme, and it also has following feature:
(5) 5 ' and 3 ' binding sequence is respectively 7-12 Nucleotide;
(6) 5 ' are connected by GGCTAGCTACAACGA catalysis sequence with 3 ' binding sequence,
(7) form by 29～39 Nucleotide;
(8) cutting is arranged in the purine of LMP1 mRNA and the connecting key between pyrimidine specifically.
Oligonucleotide of the present invention can also be the ribozyme with its feature.
Oligonucleotide of the present invention is the antisense oligonucleotide with its feature.
Oligonucleotide of the present invention can also have its feature, suppresses the interference type RNA (RNAi) of EBV LMP1 genetic expression; These interference types RNA is two key RNA that 21～25 Nucleotide are formed, and at 3 of every chain ' end 2 outstanding Nucleotide is arranged; Double-stranded G+C content is 30～52%; To 19 positions, be the A/U pairing at 15 on the positive-sense strand; No intramolecularly tumor-necrosis factor glycoproteins.
Above-mentioned described various oligonucleotide can be used for preparing multiple inhibition nasopharyngeal carcinoma growth pharmaceutical composition.Can contain various pharmaceutically acceptable vehicle, assistant agent in the described pharmaceutical composition.In the described pharmaceutical composition, can contain one or more described oligonucleotide.Such as, can contain two kinds, three kinds, four kinds, the oligonucleotide provided by the invention more than five kinds or five kinds.
Can prepare the medicine that strengthens nasopharyngeal carcinoma radiotherapy susceptibility with oligonucleotide of the present invention as active substance.
Oligonucleotide provided by the invention for animal or human's dosage is: per kilogram of body weight 1～50mg.
Oligonucleotide refers to and contains Nucleotide (Yeast Nucleic Acid, thymus nucleic acid or both) molecule, these Nucleotide by 5 ' or 3 ' end or 5 ' or 2 ' end connect, used Nucleotide can be natural, also can be the analogue of chemosynthesis, these analogues can form pairing with natural acid.For example, azepine (Aza) and denitrogenation (deaza) pyrimidine analogue of mixing, azepine (aza) and denitrogenation mix (deaza) purine and other heterocyclic base analogue.These base analogues are included in one or more carbon nitrogen-atoms in pyrimidine or the purine by replacements such as oxygen, sulphur, selenium, phosphorus.
In order to use oligonucleotide effectively in vivo, it is carried out chemically modified can increase the stability that oligonucleotide is antiacid and antienzyme is degraded significantly, for example, 2 '-O-methyl, 2 '-O-allyl group, delivery ceremony nucleic acid (Locked nucleicacids, LNA) and peptide chain nucleic acid (peptide nucleic acids, PNA).
Oligonucleotide provided by the present invention can be modified with the mode of chemistry.Described modification is on molecular level the natural acid molecular structure to be carried out a place or many places chemically modified.
Comprise following chemically modified:
1) to base, glycosyl, the modification of the phosphoric acid ester bond between the Nucleotide, and increase some substituting groups, for example, diamines (diamine), cholesterol or other lipophilic groups because of.
Separate the nucleic acid molecule of enzyme liberating in the anti-nucleic acid.This antienzyme characteristic can be modified by number of chemical and be obtained, and comprises 2 '-O-methyl acid phosphate diester linkage,, 2 '-O-methane-n (O-methane), 2 '-fluorine, heterozygosis connecting key, the modification of other trunk structure.In addition, these modify the modification that also comprises base, for example, and methylated base, methylolated base etc.
Modification nucleotide also comprises the modification to glycosyl, for example at the Yeast Nucleic Acid or the thymus nucleic acid of 2 ' 3 replacement.Connecting key between ribosyl and the mononucleotide is called the trunk structure of nucleic acid.The modification of trunk structure is comprised that connecting key and other can enhanced stability and the modification of affinity, for example, to the modification of sugared structure.Concrete example has, and when being reverse with respect to natural dextrorotatory isomer sugar, can use left-handed (L-) isomer ribodesose in base; Or with 2 ' of glycosyl-hydroxyl because of, 2 '-halogens, 2 '-O-alkyl, 2 '-O-alkyl-n (O-alkyl) replaces; Perhaps use following connecting key: 2 '-O-methyl acid phosphate diester linkage, 2 '-O-ethyl, 2 '-O-propyl group, butyl, 2 '-O-alkyl-n (alkyl), 2 '-methoxyethoxy, 2 '-fluorine; 2 '-deoxidation erythropentofuranosyl; 3 '-O-methyl; The isobutyl-oligonucleotide, 2 '-O-(CH
2CH
2)
xCH
3And Butyne connecting key.The present invention preferably 2 '-O-methyl modifies.Oligonucleotide of the present invention can have part to be modified, or all modifies, or the combination of different modifying.
2) terminal protection: the end of molecule add protecting group because of, to prevent the degraded of molecule.This protection can be 5 ' end, also can be 3 ' end, or two ends is all protected.
Oligonucleotide can be by terminal modifiedly obtaining this antienzyme characteristic to its 5 ' end and 3 '.These end modified comprising: oppositely connect base, dideoxy nucleotide, METHAPHOSPHORIC ACID base, alkyl, aryl, cordycepin, cytosinearabanoside, 2 '-methoxyl group, oxyethyl group Nucleotide, phosphorothioate connecting key, 3 '-O-methyl base, fluorescein, peptide bond connects, two pyridyls, cholesterol, vitamin H, acridine, rhodamine psoralen, glyceryl, the butanols base, butyl, alcohol radical and 3 '-O-alkyl.The preferred butanols based structures of the present invention is OH-CH
2CH
2CH
2CH
2-.
The present invention preferably connecting key of modification property oligonucleotide is achirality (achiral), can contain at least 3～8 successive achirality connecting keys, is preferably 7～12 achirality successive connecting keys; Most preferably whole connecting keys is achiral.In some cases, achirality connecting key and chirality connectivity can alternately exist, and for example, connect a chirality connecting key behind two continuous achirality connecting keys, again with two continuous achirality connecting keys; Or chirality connecting key of three successive achirality keyed jointings; Or four successive achirality connecting keys are followed two chirality connecting keys etc.The achirality connecting key includes, but not limited to phosphodiester bond, the thiophosphoric acid diester linkage.
Phosphodiester bond can be 3 ' to 3 ', 5 ' to 2 ' or 5 ' to 5 ' or above combinations of directions.The modification of this connecting key can be intramolecular (single or repetition), also can be the end at RNA or DNA.
Using DNAzyme is a kind of novel gene inhibition technology, and it has the chemical stability of antisense oligonucleotide, have the function of the catalyze cleavage RNA of ribozyme again simultaneously, and its synthetic expense is very low.Based on these unique advantages, this technology has been widely used in the body and external gene regulating (pharmacol.Rev.52 (3) such as Sun: 325-347).
Therefore, the invention provides the modification DNAzyme that can suppress EBV genetic expression.The structure of this DNAzyme comprises: (1) catalysis gene, its nucleotides sequence is classified as: GGCTAGCTACAACGA.(2) be positioned at catalysis gene 5 ' end binding sequence; (3) be positioned at the binding sequence that catalysis gene 3 ' is held.DNAzyme is by these two binding sequences, and the RNA with EBV genetic expression combines specifically.Thereby induce cutting at purine and pyrimidine binding site to target RNA by the catalysis gene.
The functional effect of pharmaceutical composition provided by the present invention can be measured with method well known in the art.These methods comprise that body is interior and external, for example, analyze pharmaceutical composition to the influence of expression of target gene and the influence of pair cell phenotype in cell culture system; In model and the clinical trial, measure the biological effect of pharmaceutical composition in vivo, comprise drug effect, pharmacology, medicament, toxicity etc.The body inner model comprises the disease animal model and the intact animal of foundation.
Pharmaceutical composition provided by the invention contains more than one the oligonucleotide that suppresses EBV genetic expression.According to the quantity of oligonucleotide, type, target gene etc., oligonucleotide has a multiple different array mode in the pharmaceutical composition.
Can only contain single oligonucleotide in the pharmaceutical composition, its target gene can be the EBV gene, also can be host's genes involved.
The oligonucleotide that can contain more than one in the pharmaceutical composition, the kind of oligonucleotide can reach below 100 kinds, preferably 2～20 kinds.
Oligonucleotide in the pharmaceutical composition can be one type, for example is selected from DNAzyme, antisense oligonucleotide, or ribozyme respectively; Or interferential RNA.
Also can comprise dissimilar oligonucleotide in the pharmaceutical composition, for example, the combination of DNAzyme and RNA interfering, the combination of DNAzyme and antisense oligonucleotide etc.
The present invention includes pharmaceutical composition and contain at least a oligonucleotide provided by the present invention.In some pharmaceutical composition, can contain oligonucleotide different below 100 kinds, for example 2,3,4,5,6,7,8,9,10,15,20,30,40,50,75 kinds.In some pharmaceutical composition, oligonucleotide is an antisense oligonucleotide, ribozyme, interferential RNA, DNAzyme, or above combination.In some pharmaceutical composition, one or more oligonucleotide are modification property oligonucleotide.In some compound, contain more than one antisense oligonucleotide, it can be the pharmaceutical composition of partly or entirely modifying.Also contain in the pharmaceutical composition and be suitable for medicinal vehicle and other assistant agent.
Target gene of the present invention can be single, also can be the combination of several genes.The pharmaceutical composition of target several genes can be regulated and control simultaneously to different signal transduction paths, will be more more effective than the method for target term single gene.In addition, because the amount of each oligonucleotide in a plurality of oligonucleotide that use is relatively low, therefore can reduce toxigenous possibility greatly.The oligonucleotide that the present invention uses is for modification property, and is therefore more stable than natural oligonucleotide, and compares with modifying method commonly used at present, for example, the thiophosphoric acid diester linkage, the toxicity of oligonucleotide of the present invention is lower.
Pharmaceutical composition of the present invention can adopt parenteral, oral or partial administering mode.
Pharmaceutical composition of the present invention can be prepared by technology well known in the art.The selection of pharmaceutical carrier can be according to preparation type and route of administration, for example, intravenous injection, oral, the part, sprays, suppository, non-enteron aisle, or bone marrow injection etc.Vehicle can contain several pharmaceutical carriers.In addition, contained stablizer in the pharmaceutical composition, sanitas and other composition are generally 0.5%～2% of pharmaceutical composition weight.Those skilled in the art can determine suitable administering mode and delivery system.
Pharmaceutical composition of the present invention can be made different formulations on request, comprises liquid, tablet, and pill, granular, powder, ointment, emulsion, injection or suppository.Any medicine acceptable medium and assistant agent all can use, for example, water, glycerine, oils, ethanol, seasonings, sanitas, food dye etc. are used for liquid preparation, starch, sugar, thinner, granule, lubricant, tackiness agent, dispersion agent etc. are used to prepare solid preparation.
Injection preparation is the oligonucleoside aqueous acid, and solvent is water or the physiological saline that meets medicinal standard.Injection liquid can also contain suitable liquid medium, suspension agent and the preparation that can regulate osmotic pressure, sanitas etc.Actual fabrication method and program are known for a those skilled in the art.
Local application's formulation can be made emulsion, dressing, gel, washing lotion, ointment, liquid etc.Can add tensio-active agent, penetrate with the deep layer that increases medicine.These tensio-active agents comprise natural, also can be chemosynthesis, for example different third myristate.
The preparation of aerosol can be adopted and oligonucleotide is dissolved in or is suspended in hair spray or hair spray and the solvent, and for example ethanol is as solvent.In local application's formulation and aerosol, drug ratios (oligonucleotide) is generally 0.001%～40% of gross weight.
The preparation of suppository can mix oligonucleotide and make with lipid medium, theobroma oil for example, cocoa cream, glycerine, gelatin or polyoxyethylene glycol.
Oligonucleotide can also be made liposome, to increase the oligonucleotide transformation period in vivo.Used lipid comprises, but be not limited to, Cardio1ipin dimyristoyl, dipalmifoyl, dioleoyl phosphatidyl choline, phospha tidyl gly cerol, palmitoyloleotl phosphatidyl choline or phosphatidylglyceroe, phosphatidi acid cysophosphatidic acid, phosphatidyl serine, phosphatidye inositol, cholesterol.
The dosage of pharmaceutical composition of the present invention is the described oligonucleotide of per kilogram of body weight 0.01～5mg, can be respectively 0.01,0.05,0.1,0.5,1 as per kilogram of body weight, the described oligonucleotide of 5mg.Preferred dosage is per kilogram 0.01mg～10mg, or per kilogram weighs 0.01～1mg, or 0.1～1mg.In some cases, unitary dose can be per kilogram of body weight 0.01～100mg.
When implementing the tumour inner injecting and administering, but dose unit of administration in per 1～10 day, or administration every day, every day 1～10 dose unit, up to healing, or symptom is alleviated.In some cases, patient is by 2～3 dose units of doctor's advice medication every day.In some cases, administration in 1-24 hour before radiotherapy, dosage is every day 1～4 time.
Target EBV-LMP1 oligonucleotide provided by the invention is applied to clinical patients, finds that target EBV-LMP1 oligonucleotide can increase the susceptibility of nasopharyngeal carcinoma patient to radiotherapy, accelerates tumour and dwindles speed.Simultaneously according to RTOG acute radiation injury grade scale, by in the process of the test to the observation of its index, show that oligonucleotide does not all increase the damage of hemopoietic system, skin, mucous membrane, sialisterium and hepatic and renal function to the patient.Show that the oligonucleotide of target EBV-LMP1 not only may become the New Policy of target gene therapy, may develop into a kind of radiotherapeutic sensitizer clear and definite target, novel that has simultaneously.
Description of drawings
DNAzyme that Fig. 1 .2 '-0-methyl modification/3 '-butanols tailing is modified and the contrast of the DNAzyme result of unmodified;
Fig. 2. the cell death inducing of DNAzyme dose-dependently in the CNE1-LMP1 cell also strengthens radiation inductive apoptosis;
Fig. 3. DNAzyme is cell death inducing and enhancing radiation inductive apoptosis in the CNE1-LMP1 cell;
Fig. 4. the DNAzyme DZ1 of target LMP1 suppresses the increment of CNE1-LMP1.The result represents with mean value plus-minus standard deviation from three independent experiments;
Fig. 5. DNAzyme acts on separately or unites use back p53 with radiation, and survivin expresses in the CNE1-LMP1 cell and obviously is suppressed;
Fig. 6. DNAzyme use separately or unite use with radiation after can significantly suppress transplanted tumor in nude mice.A specificity DNAzyme is used separately or is united the growth that can significantly suppress transplanted tumor after the use with radiation.The tumor weight that the B treatment was downcut after 24 hours;
Fig. 7 .A immunohistochemical analysis LMP1 and the expression of Bc1-2 in transplanted tumor in nude mice.LMP1 and Bc1-2 antibody incubation are used in section respectively.There is not an anti-negative contrast of conduct of hatching processing.(200X) B HE dyeing is carried out pathology detections (200X) to different treatment group tumor tissues;
Embodiment
Embodiment
Implement the chemosynthesis of a modification property DNAzyme
Building-up process is finished by computer-controlled ABI synthesizer.The different pipelines of differential responses component sequencing ground warp enter reaction chamber (reaction column).
1. the foundation of solid state reaction
Building-up reactions is carried out in leaning on containing the covalently bound monomeric reaction of 3 '-butanols base.
2. chain extension reaction
The synthetic Cyano-ethyl-phosphoramidite chemistry that adopts carries out.Each monomer all contains 2 '-0-methyl.Comprise each reaction time: reactive group go the protection; In conjunction with new monomer; Protect non-reactive group; Oxidizing reaction changes into triphosphate (triphophate) with triphosphoric acid thing (triphosphite).With this process, each cycle is introduced a new monomer.
3. protective reaction
After finishing last mononucleotide reaction, the use aqua ammonia discharges reaction product and removes blocking group from reaction is leant on, remove solid matter by vacuum filtration then.
4. aftertreatment
Reaction product is removed aqua ammonia by vacuum-drying, then through the HPLC purifying, obtains＞99% pure product.
The specific activity of enforcement two modification property DNAzyme and unmodified DNAzyme
Obtain higher biological activity in order to prove that modification property DNAzyme increases because of its stability with LMP-1 mRNA bonding force, experiment is extracted the present invention's modification property DNAzyme Dz1 (5 ' gcaaaggaaGGCTAGCTACAACGAagaggacaa3 ') and unmodified DNAzyme Dz1 difference transfection CNE1LMP1 cell total protein and is carried out Western Blot analysis.From Fig. 1 as seen, modify and 3 '-butanols tailing through 2 '-0-methyl, the activity that suppresses the LMP-1 expression at the DNAzyme of LMP-1 significantly improves (10 times).
Implementing three modification property DNAzyme modifies the property DNAzyme and strengthens the susceptibility of nasopharyngeal carcinoma cell to radiotherapy in external knurl and the radio therapy sensitization effect of pressing down
The result shows, compare with independent radiotherapy or independent DNAzyme processing, DNAzyme Dz1 provided by the invention and radiotherapy combined utilization can significantly strengthen the apoptosis of Induced by Radioactive Ray, and have dose-dependently, when 5Gy, 80% cell generation apoptosis (Fig. 2).Select radiotherapy dosage and the active deoxidizing ribozyme combined utilization of 2Gy, find to compare with independent DNAzyme or radiotherapy, 3 active deoxidizing ribozymes all can significantly strengthen the susceptibility (Fig. 3) of cell to radiotherapy.Modification property DNAzyme and radiotherapy combined utilization suppress nasopharyngeal carcinoma cell propagation
The result shows, compares with control group and independent DNAzyme or combination radiotherapy group, and DNAzyme and radiotherapy combination treatment group significantly suppress the propagation (Fig. 4) of nasopharyngeal carcinoma cell.
Modification property DNAzyme and radiotherapy combined utilization suppress cell cycle of human nasopharyngeal carcinoma and advance
The radiotherapy opposing is advanced closely related with the cell cycle.The influence that 3 active deoxidizing ribozymes of applying flow cytometry observation and radiotherapy combined utilization were advanced to the cell cycle of nasopharyngeal carcinoma cell CNE1-LMP1.The result shows that the active deoxidizing ribozyme can cause the cell cycle change of nasopharyngeal carcinoma cell CNE1-LMP1, compares with independent radiotherapy, S phase cell is increased, and G2/M phase cell reduces (table 1).
Table 1 active deoxidizing ribozyme is to the * that influences of nasopharyngeal carcinoma cell CNE1-LMP1 cell cycle
* numeral cell shared per-cent in the different cell cycles among the figure.
DNAzyme and radiotherapy combined utilization suppress the expression of p53 and Survivin
Studies show that LMP1 participates in the expression of the machine-processed LMP1 of being of possibility of radiotherapy opposing by trans-activation p53 and Survivin, and then participate in the generation of radiotherapy antagonism.Therefore, use the influence of western blotting detection DNAzyme and radiotherapy combination treatment to the expression of p53 and Survivin, found that compared with the control the expression (Fig. 5) that DNAzyme after the present invention modifies and radiotherapy combined utilization can be reduced p53 and Survivin.
Implement four modification property DNAzyme and press down knurl and radio therapy sensitization effect in the animal model in vivo
Set up nasopharyngeal carcinoma transplanted tumor in nude mice model, observe the influence of DNAzyme and radiotherapy combined utilization the nasopharyngeal carcinoma transplanted tumor in nude mice.The result shows that compared with the control, DNAzyme and radiotherapy combined utilization can significantly suppress the growth (Fig. 6) of transplanted tumor in nude mice.
Tumor tissues is carried out pathological observation.The HE coloration result shows that in control group and independent combination radiotherapy group, cellular form is not seen change; And in modification property DNAzyme treatment group and modification property DNAzyme and radiotherapy combination treatment group, it is downright bad that the part tumour cell occurs; Illustrate that DNAzyme can strengthen tumour cell in vivo to the susceptibility of radiotherapy (Fig. 7-B).The expression of LMP1 and Bc1-2 in the immunohistochemical methods detection tumor biopsy, combination radiotherapy group and contrast add combination radiotherapy group separately, and LMP1 and Bc1-2 are strong positive; And modification property DNAzyme and radiotherapy combination treatment group, and all significantly downward modulations of the expression of LMP1 and Bc1-2 (Fig. 7-A).
Implement the clinical study of five modification property DNAzyme radio therapy sensitizations
Can v be applied to EBV-LMP1 male nasopharyngeal carcinoma patient clinically with the DNAzyme of target LMP1, observe target EBV-LMP1 DNAzyme and strengthen the susceptibility of nasopharyngeal carcinoma patient to radiotherapy, observes its toxic side effect simultaneously.
The research object inclusion criteria
From year November Xiangya Hospital, Central-South China Univ.'s oncology in March, 2007-2007 pathology be diagnosed as first nasopharyngeal carcinoma patient 20 examples of controlling of low differentiated squamous-cell carcinomas;
2. between year, the men and women does not limit at 20-65 the age;
3. clinical diagnosis does not have distant metastasis;
4. immunohistochemical methods shows the patient of LMP1 positive expression;
5. sign Informed Consent Form voluntarily;
6. do not need in the radiotherapy in conjunction with chemotherapy and other special treatment persons;
7. Ka Shi (KPS) scoring＞70, and no cardiovascular, liver kidney and hemopoietic system etc. are primary disease seriously, can finish radiotherapy smoothly.
Research and design
The 20 routine nasopharyngeal cancer patients that will meet into the group standard are divided into two groups at random by double-blind method, test group (A group, radiotherapy adds target EBV-LMP1 DNAzyme) and control group (B group, radiotherapy adds physiological saline), two groups of radiation therapy methods are identical, use the external conventional planning treatment of irregular lead block, nasopharynx irradiation dose DT70～76Gy, 7～7.5 weeks finished, positive lymphonodi cervicales DT66～70Gy, and 6～7 weeks finished; The negative prophylactic irradiation DT50～56Gy of lymphonodi cervicales, 5～6 weeks finished.A group on every Mondays, preceding 2 hours of four radiotherapies, locally injected into tumor DNAzyme 0.1ml, 120mg/ml, totally 10 times.The B group is given physiological saline 0.1ml, and other treatment and processing two groups of A, B are identical.Endoscope is observed the tumour size variation down weekly; By before importing radiotherapy, 4 MRI images of check in March are accurately measured gross tumor volumes after radiotherapy 50Gy, radiotherapy end and the radiotherapy; Adopt the objective therapeutic evaluation criterion calculation of noumenal tumour WHO CR, PR; And observe toxic side effects in its use according to RTOG acute radiation injury grade scale.
Result of study
High conformity between test group and control group patient's general data packet group illustrates that the homogeneity of test group and control group, harmony are all higher.Carry out the parallel t check of conversion (RTR value) of rate according to the body of knurl shown in MRI volume (cm3), the result shows that RTR3 compares that the P value is 0.04 between test group and control group group before check and the radiotherapy after March, significant difference is arranged, think that target EBV-LMP1 DNAzyme can strengthen the radiation sensitivity (table 2) of LMP1 male nasopharyngeal cancer patient.Two groups CR, PR compare, and significant difference, there was no significant difference are not temporarily found in P value=0.092 because of routine number is very few.The damage there was no significant difference of hemopoietic system, skin, mucous membrane, sialisterium, hepatic and renal function between two groups.Target EBV-LMP1 DNAzyme has strengthened the susceptibility of LMP1 male nasopharyngeal carcinoma patient to radiotherapy, has improved tumor regression speed.In the radiotherapy process, can not increase nasopharyngeal carcinoma radiotherapy patient's acute radiation injury, have good security.
Table 2: the different treatment group is put the t assay of a RTR for a long time
In sum, target EBV-LMP1 DNAzyme provided by the invention is applied to clinical patients, finds that target EBV-LMP1 DNAzyme can increase the susceptibility of nasopharyngeal carcinoma patient to radiotherapy, accelerates tumour and dwindles speed.Simultaneously according to RTOG acute radiation injury grade scale, by in the process of the test to the observation of its index, show that DNAzyme does not all increase the damage of hemopoietic system, skin, mucous membrane, sialisterium and hepatic and renal function to the patient.Show that the DNAzyme of target EBV-LMP1 not only may become the New Policy of target gene therapy, may develop into a kind of radiotherapeutic sensitizer clear and definite target, novel that has simultaneously.
Claims (14)
1. oligonucleotide has following feature:
(1) by its binding sequence and EBV LMP1 gene complementation;
(2) form by 11～70 Nucleotide, from 5 ' to 3 ' direction, connect by the phosphoric acid ester bond;
(3) on ribose 2 ' position, have the methoxyl group substituted radical;
(4) at 3 ' the terminal butanols group that connects.
2. oligonucleotide according to claim 1, the connecting key that it is characterized in that oligonucleotide are to contain at least 3～8 successive achirality connecting keys, are preferably 7～12 achirality successive connecting keys; Most preferably whole connecting keys is achiral.
3. oligonucleotide according to claim 1, the connecting key that it is characterized in that oligonucleotide are that achirality connecting key and chirality connectivity can alternately exist.
4. oligonucleotide according to claim 1 and 2 is characterized in that described oligonucleotide is a DNAzyme, and it also has following feature:
(5) 5 ' and 3 ' binding sequence is respectively 7-12 Nucleotide;
(6) 5 ' are connected by GGCTAGCTACAACGA catalysis sequence with 3 ' binding sequence,
(7) form by 29～39 Nucleotide;
(8) cutting is arranged in the purine of LMP1mRNA and the connecting key between pyrimidine specifically.
5. oligonucleotide according to claim 1 and 2 is characterized in that described oligonucleotide is the ribozyme with its feature.
6. oligonucleotide according to claim 1 and 2 is characterized in that described oligonucleotide is the antisense oligonucleotide with its feature.
7. oligonucleotide according to claim 1 and 2 is characterized in that described oligonucleotide has its feature, suppresses the interference type RNA (RNAi) of EBV LMP1 genetic expression; These interference types RNA is two key RNA that 21～25 Nucleotide are formed, and at 3 of every chain ' end 2 outstanding Nucleotide is arranged; Double-stranded G+C content is 30～52%; To 19 positions, be the A/U pairing at 15 on the positive-sense strand; No intramolecularly tumor-necrosis factor glycoproteins.
8. suppress nasopharyngeal carcinoma growth medicine, contain in the described oligonucleotide of claim 1 to 7 one or more; Or contain one or more and various pharmaceutically acceptable vehicle, assistant agent in the described oligonucleotide of claim 1 to 7.
9. strengthen the medicine of nasopharyngeal carcinoma radiotherapy susceptibility, contain in the described oligonucleotide of claim 1 to 7 one or more, or contain one or more and various pharmaceutically acceptable vehicle, assistant agent in the described oligonucleotide of claim 1 to 7.
10. according to Claim 8 or 9 described medicines, it is characterized in that the oligonucleotide that contains is the 2-20 kind.
11. according to Claim 8 or 9 described medicines, it is characterized in that also containing in the said oligonucleotide oligonucleotide of unmodified.
12. according to Claim 8 or 9 described medicines, it is characterized in that by a dose unit be the described oligonucleotide administration of per kilogram of body weight 0.01～5mg.
13. medicine according to claim 12, when it is characterized in that implementing interior injection of tumour or tumor by local spray delivery, but dose unit of administration in per 1～10 day; Or administration every day, every day 1～10 dose unit, preferred 2-3 dose unit.
14. medicine according to claim 12 is characterized in that administration in 1-24 hour before radiotherapy, dosage is every day 1～4 time.
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| CN200810143058A CN101712710A (en) | 2008-10-08 | 2008-10-08 | LMP1targeted oligonucleotide and application thereof serving as radiotherapeutic sensitizer |
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810143058A CN101712710A (en) | 2008-10-08 | 2008-10-08 | LMP1targeted oligonucleotide and application thereof serving as radiotherapeutic sensitizer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940783A (en) * | 2010-08-03 | 2011-01-12 | 孙仑泉 | Application of DNAzyme in preparing medicines for enhancing tumor chemotherapy sensitivity |
| CN102552944A (en) * | 2012-01-18 | 2012-07-11 | 中南大学 | Nasopharyngeal carcinoma targeted magnetic resonance contrast agent and preparation method thereof |
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| CN101147801A (en) * | 2006-09-18 | 2008-03-26 | 中南大学 | Application of dexoyribozyme of target EBV-LMP1 in preparing medicine for treating EBV corresponding solid tumor |
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| CN101147801A (en) * | 2006-09-18 | 2008-03-26 | 中南大学 | Application of dexoyribozyme of target EBV-LMP1 in preparing medicine for treating EBV corresponding solid tumor |
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| LU ZX，等: "Effect of EBV LMP 1 targeted DNAzymes on cell proliferation and apoptosis", 《CANCER GENE THERAPY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940783A (en) * | 2010-08-03 | 2011-01-12 | 孙仑泉 | Application of DNAzyme in preparing medicines for enhancing tumor chemotherapy sensitivity |
| CN102552944A (en) * | 2012-01-18 | 2012-07-11 | 中南大学 | Nasopharyngeal carcinoma targeted magnetic resonance contrast agent and preparation method thereof |
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