CN101338303B - Adenovirus labeled with radionuclide - Google Patents
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- CN101338303B CN101338303B CN 200810304321 CN200810304321A CN101338303B CN 101338303 B CN101338303 B CN 101338303B CN 200810304321 CN200810304321 CN 200810304321 CN 200810304321 A CN200810304321 A CN 200810304321A CN 101338303 B CN101338303 B CN 101338303B
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- radioisotope labeling
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- 241000701161 unidentified adenovirus Species 0.000 title claims abstract description 99
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Abstract
The invention relates to adenovirus marked by radionuclide, belonging to the field of tumor treatment. The radionuclide-labeled glandViruses are modified by covalent or chelating radionuclides on the adenovirus capsid protein. The radionuclide is selected125I、131I、32P、188Re、186Re、90At least one of Y. The adenovirus marked by the radionuclide can play a better anti-tumor role, and provides a new choice for the field.
Description
Technical field
The present invention relates to the adenovirus of radioisotope labeling, relate to the oncotherapy field.
Background technology
Adenovirus is a kind of nonencapsulated double-stranded DNA virus, and genome is about 36kb, and core is by the distrand DNA chain, and nucleoprotein TP and DNA polymerase constitute, and capsid is 20 body structures of rule, the about 80-110nm of diameter.Capsid contains 240 six conjuncted, 12 5-linked bodies and 12 ciliums, in addition also has some other small protein, as VI, VIII, IX etc.Six conjuncted be the major protein that forms 20 gores of viral capsid, 12 tops are 5 5-linked body subunits and 3 mixtures that cilia protein constitutes, 12 ciliums are that substrate is stretched out by the capsid surface with the 5-linked body protein, the cilium top forms the cephalomere district.The cephalomere district of 5-linked body and cilium can combine with the virus receptor of cell surface, plays important effect in the virus infected cell process.
Also has the report that much adenovirus is used in the tumor biotherapy in recent years, as use replication-defective adenoviral to carry the encoding gene of some anti-tumor factors as carrier, express these factors again after entering in the body, expectation can be played long lasting antitumor action, to overcome the defective of direct these anti-tumor factors of infusion.
The oncolytic adenovirus is as a kind of novel means of treatment tumour, and at present, its research has obtained heartening achievement
[1-2], because can in tumour cell, duplicate, the cracking tumour cell, become this area one big focus.Also the someone develops the oncolytic adenovirus that can express anti-tumor factor in vivo simultaneously, with the performance double effects.Such as disclosing a species specific oncolytic adenovirus among the Chinese ZL200410046237.x, called after adenovirus KH901 also discloses its gene order of its preparation method simultaneously.Adenovirus KH901 both can duplicate in tumour cell, the cracking tumour cell, and can stimulate the antitumor action of body at tumor locus expression of GM-CSF.At present, this product has entered the clinical study stage at present.
It is generally acknowledged outside traditional tumor chemoradiotherapy, can use the gene therapy means as effectively replenishing, expect both can by different mechanism and approach acting in conjunction to reach antineoplastic purpose.As Spear
2Deng the ionizing rays that studies show that can not change the virus replication ability and the anti-tumor activity of enhanced virus hrR3.Mice lung cancer cell after experiment confirm adopts adenovirus carrier infection gammairradiation is also arranged, and the lu gene encoding production is the dose-dependently amplification in the cell, and growth of tumour cell obviously is suppressed
3Bradley
4Deng having confirmed that radiotherapy unites the R3616 that deletes ICP34.5 and treat, can suppress tumor growth better, thereby improve patient's survival rate.There is no the report of radioisotope labeling adenovirus and application thereof both at home and abroad.
Summary of the invention
Technical problem underlying to be solved by this invention is to improve adenovirus anti-tumor in vivo activity.The technical scheme that solves this technical problem provides a kind of adenovirus of being modified by radionuclide, is modified with radionuclide on the capsid protein of this adenovirus.
Wherein, above-mentioned radionuclide is
125I,
131I,
32P,
188Re,
186Re,
90At least a among the Y.
Wherein, above-mentioned adenovirus is the oncolytic adenovirus.Preferably, above-mentioned oncolytic adenovirus is adenovirus KH901.
Wherein, the radionuclide of modifying on above-mentioned each adenovirus is 5.0 * 10
-3～2.0 * 10
-2Bq.Preferably, the radionuclide of modifying on above-mentioned each adenovirus is 8.3 * 10
-3～9.3 * 10
-3Bq.
Second technical problem that the present invention will solve provided a kind of method for preparing the adenovirus of above-mentioned radionuclide modification.This method may further comprise the steps:
Preparing concentration is 1 * 10
8～1 * 10
10The adenovirus solution of VP/ml adds 1 * 10 in per then 100 μ l adenovirus solution
5～1 * 10
7The radionuclide of Bq, per again 100 μ l add N-bromosuccinimide (NBS) 1～100 μ g, add phosphate buffered saline buffer under the room temperature and regulate pH value to 7～7.6.Reacted 2～5 minutes, and added 2% human serum albumin (HSA) solution termination reaction again.Preferable methods is: preparing concentration is 8 * 10
9The adenovirus KH901 of VP/ml, per then 100 μ l adenovirus solution add 7.4MBq
131I/
125I and 25 μ g NBS add the pH value and are the phosphate buffer 1 20 μ l of 7.5 0.05mol/L again, react 3min under the room temperature, add the HSA solution termination reaction of 10 μ l 2% again.
The 3rd technical problem to be solved by this invention provided the purposes of adenovirus in the preparation antineoplastic pharmaceutical compositions that above-mentioned radionuclide is modified.
The 4th technical problem to be solved by this invention provided a kind of antineoplastic pharmaceutical compositions.This antineoplastic pharmaceutical compositions is to add pharmaceutically acceptable complementary composition by the adenovirus that above-mentioned radionuclide is modified as main active ingredient to be prepared from.
According to a first aspect of the invention, can modify the capsid protein of adenovirus, close as covalent modification or huge legendary turtle with radionuclide.Can make a certain amount of radionuclide on the capsid mark of each adenovirus behind the mark, to reach the effect that can suppress tumor growth in vivo.Amount according to concrete tumour kind and the adenovirus that will use can have a bigger domain of walker to the labelled amount on each adenovirus, with combining preferably of the effect of the effect that reaches adenovirus and radionuclide.For the oncolytic adenovirus, the radionuclide of modifying on each adenovirus is 5.0 * 10
-3～2.0 * 10
-2Bq is a scope preferably.Radionuclide is can be by chemical bond covalently bound or be chelated to polypeptide or the enterprising row labels of proteinic amino-acid residue.But make labelled amount reach requirement of the present invention and can not destroy the structure of adenovirus and activity (labelled amount can calculate according to the amount of the virus that is added and the amount by the measured radionuclide of γ calculating instrument).Chelating can use adenovirus capsid Argine Monohydrochloride sulfydryl huge legendary turtle metal radionuclide, and method is (as SnCl with reductive agent
2, V-Brite B, stannous tartrate) but the cystine linkage of adenovirus capsid Argine Monohydrochloride is reduced to the sulfydryl of huge legendary turtle metal radionuclide, to finish mark; Perhaps by the metal radionuclide that closes the difunctional intercalating agent huge legendary turtle of amino-acid residue link coupled.The chelation group energy mortise nucleic ion of the specific stereochemical structure of bifunctional chelating agent, the active functional group group of containing can be connected with proteinic amino acid fragment by covalent linkage, indirect like this radionuclide is connected on the protein molecular, this method is also smaller to the activity influence of marker, and the body internal stability is also relatively good.The method that iodine is modified can be used existing iodine labeling method of protein (as: chloramine-t method, NBS method), and the site of covalent modification is mainly the casein amino acid residue on the capsid protein, and main decorating site is the hydrogen atom that replaces on the tyrosine phenyl ring.Certainly, provide a kind of radioiodine to modify the method for oncolytic adenovirus in the present invention, the experiment proved that to reach requirement.Radionuclide is meant that atomic energy spontaneously launches some special ray and be transformed into the unstable nucleic of another kind of nucleic.The kind of operable common radionuclide is
125I,
131I,
32P,
188Re,
186Re,
90At least a among the Y, the present invention with
125I/
131I is the example of model as research.
Adorned adenovirus can be common adenovirus in the adenovirus of radioisotope labeling of the present invention, also can be the oncolytic adenovirus.Radionuclide can be combined the restraining effect of tumour and the antitumor action of oncolytic adenovirus, reach better effect.Also can modify the adenovirus carrier that carries the tumor protein p53 gene separately in addition, so just the effect of tumor protein p53 and the effect of radionuclide can be combined, play better antitumous effect.The preferred this oncolytic adenovirus that can express anti-tumor factor in vivo of adenovirus KH901.
Beneficial effect of the present invention is: penetrating property of the present invention nucleic
131The adenovirus of I covalent modification not only self activity of virus is not affected, can express anti-tumor factor by normal replication adenovirus oncolytic justacrine, and the experiment in vivo and vitro result also shows: viral oncolytic therapy combines with the effect of radionuclide, can improve lethal effect greatly to tumour cell, can suppress intravital tumor growth better, well play synergy.Can play better effect, reduce misery and inconvenience that patient injected and carried out repeatedly radiotherapy repeatedly simultaneously, reduce the treatment cost, improve quality of life, also make treatment procedure easier, improve therapeutic efficiency.For new mode has been opened up in oncotherapy.
Description of drawings
Fig. 1 is for adding volume and the mark rate relation of PBS
Fig. 2 is a tumor bearing nude mice
131I-KH901 video picture (anteposition, 2h, 6h, 12h, 24h).
Fig. 3 is tumor bearing nude mice Na
131I video picture (anteposition, 2h, 6h, 12h, 24h).
The present invention will be described in detail by embodiment below in conjunction with accompanying drawing
Embodiment
The preparation of the adenovirus that embodiment one radionuclide of the present invention is modified
Generally speaking, but the process schematic representation of the adenovirus that preparation radionuclide of the present invention is modified is as follows:
Adenovirus-capsid protein+n radionuclide → adenovirus-capsid protein-(radionuclide) n
Radionuclide wherein can by chemical bond covalently bound (as
125I/
131I replaces the hydrogen atom of casein amino acid residue phenyl ring) or by sulfydryl chelated mineral radionuclide or by being coupled at the proteic bifunctional chelating agent chelated mineral of adenovirus capsid radionuclide, like this radionuclide is connected on the capsid protein of adenovirus surface.
Above-mentioned n is a positive number, represents the atomicity of the radionuclide of mark on each adenovirus, and its value is for to make the radionuclide amount of modifying on each adenovirus reach requirement.Because prior art is difficult to the quantity of direct detection of radioactive nucleic, thus use general represent the mode of radionuclide quantity with radioactive amount.Among the present invention the scope of comparative optimization radionuclide is 5.0 * 10 on each adenovirus in order to make
-3～2.0 * 10
-2Bq.Preferred, making the radionuclide of modifying on each adenovirus is 8.3 * 10
-3～9.3 * 10
-3Bq.
1, material is prepared:
With 24 of BALB/c mouse, the about 20g of body weight, male and female are not limit, and are divided into 8 groups, 3 every group (Sichuan University's Experimental Animal Center provides).Adenovirus KH901 provides (its its preparation method and gene order are seen Chinese patent ZL200410046237.x) by Kanghong Biotech Co., Ltd., Chengdu; Na
125I/Na
131I produces (carrier free, no reductive agent, radiochemical purity 〉=96%, pH value 7.0～8.0, γ impurity＜0.1%) by ChengDou ZhongHe GaoTong isotopes Co., Ltd, and Sephadex G-10 produces (Switzerland) for pharmacia company; N bromo-succinimide (NBS) is sigma product (USA), human serum albumin HSA (Shanghai), analytical pure; Acetone (Chengdu Xin Chuan chemical reagent limited liability company), analytical pure; γ calculating instrument (Plant No. 262), ESJ120-4 electronic analytical balance (Shenyang), eddy mixer XW-80 A (Shanghai Medical Univ), distribution collector (Shanghai).
2, preparation method:
Adopt N bromo-succinimide (NBS) as oxygenant, NBS dissolves with tri-distilled water, and concentration is 0.5mg/ml; Adjusting KH901 liquid is 8 * 10
9VP/ml; Fixing
125I/
131The consumption of I changes the consumption of KH901 and NBS, with screen best modification condition.
Add KH901 100 μ l in each reaction tubes earlier, add then
125I/
131I solution 0.5 μ l (about 7.4MBq) adds NBS solution 30,50,70,100 μ l at last respectively, adds the phosphate buffered saline buffer of an amount of 0.05mol/L under the room temperature respectively, covers the tight mouth of pipe.Use the eddy mixer mixing.After reacting about 3 minutes, every again pipe adds the HSA solution termination reaction of 10 μ l 2%.Keep other condition constant, investigate respectively again
125I/
131The consumption of I, reaction times, pH value, reaction volume are right
125I/
131The influence of I-KH901 mark rate.
The Paper Chromatography of mark rate (ratio of total active nucleus of marked product and adding) detects: stationary phase is the I of an Xinhua paper, and developping agent is an acetone: physiological saline=1: 1 (volume ratio),
125I/
131The Rf=0.0 of I-KH901～0.1, free
125I/
131The Rf=0.9 of I～1.0.Adopt same procedure, fixing
125I/
131The consumption of I and NBS, the amount that changes KH901 is respectively 25,50,75,100,130,150 μ l, reacts after 3 minutes, and mark rate is surveyed in sampling.
3, preparation method's optimization
The optimization of method and definite
A,
125I/
131The relation of I consumption, KH901 amount and NBS consumption and mark rate
125I/
131The I consumption is that 7.4MBq tense marker rate is higher; The NBS consumption reaches 25 μ g tense marker rates and reaches the highest, increases no considerable change again, and therefore, optimum reaction condition is: per 100 μ l KH901 (8 * 10
9VP/mL), 7.4MBq
125I/
131I, 25 μ g NBS.Mark rate can reach 78%
B, the influence mark rate of reaction times to mark rate prolong raising fast in time in 2min, 3min prolongs mark rate does not later in time have considerable change, and reaction 3～10min mark rate can both reach more than 75%, determines that the reaction times is 3～10min.
When c, pH value kept other condition constant to the influence of mark rate, pH increases with the pH value in 6.5～7.5 tense marker rates and improves, and was the highest to pH value 7.5 tense marker rates, increases the decline to some extent on the contrary of pH value mark rate again, and the optimal ph of determining this experiment is 7.5.
D, reaction volume influence this experiment by add a certain amount of phosphate buffered saline buffer conditioned reaction volume and pH value in reaction system to mark rate, find that to cross the hour-symbols rate not high when reaction volume, may be because the surge capability of solution be not enough, the pH value is not adjusted to the scope that helps reacting; Increase mark rate with reaction volume also increases thereupon, increases with reaction volume afterwards, and mark rate descends on the contrary, as Fig. 1.In the time of may excessively increasing owing to volume, the effective concentration of reactant reduces, and molecular interaction weakens, and mark rate reduces.It is that per 100 μ l reaction systems add 80 μ l～160 μ l that this experimental selection phosphate buffered saline buffer adds volume, and the best is 120 μ l.
The present invention is with Mather
[5]N-bromosuccinimide (NBS) method of report is as the basis, and exploitation is fit to KH901 is carried out
125I/
131The method that I modifies, the method for Que Dinging is as follows at last: preparing concentration is 1 * 10
8～1 * 10
10The adenovirus solution of VP/mL adds 1 * 10 in per then 100 μ l adenovirus solution
5～1 * 10
7The radionuclide of Bq, per again 100 μ l add NBS1～100 μ g, add phosphate buffered saline buffer under the room temperature and regulate pH value to 7～7.6.Reacted 2～5 minutes, and added 2% human serum albumin (HSA) solution termination reaction again.
Preferable methods is: when getting 100 μ l concentration is 8 * 10
9During the adenovirus KH901 of VP/mL, add 7.4MBq
131I/
125I and 25 μ g NBS add the pH value and are the phosphate buffer 1 20 μ l of 7.5 0.05mol/L again, react 3min under the room temperature, add the HSA solution termination reaction of 10 μ l 2% again.Mark rate can reach more than 78%.The radiochemicsl purity of purified back marker can reach more than 95%.This method is primarily aimed at
131I and
125I mark adenovirus, reaction system is simple, and step is easy, and the mark rate height is a kind of method of more satisfactory radioiodination adenovirus.
The purifying preparation and the vitro stability of the adenovirus of embodiment two radioisotope labelings of the present invention
(the gel filtration chromatography method of 0.5cm * 30cm) is used the modifying method preparation of embodiment one to adopt Sephadex G-10
125I/
131(the preparation respectively of the adenovirus of I mark
125I,
131The adenovirus of I mark), obtained carrying out separation and purification behind the high reaction soln of mark rate.
Saturated toward a certain amount of albumin of the interior adding of post earlier before the last sample, to eliminate the non-specific adsorption of chromatography column to adenovirus KH901.Last sample also is settled to 0.5ml with sample size, adopts Fraction Collector to collect leacheate 50 pipes continuously, and 1ml/ manages (about 15), and every pipe elutriant takes out the radiocounting (min that 10 μ l measure each pipe respectively
-1), determine the peak position of marker and free-iodine, (first peak is to get first peak
125I/
131I-KH901), through 0.22 μ m filtering with microporous membrane degerming, place 4 ℃ of preservations standby.
Record radiochemicsl purity with Paper Chromatography behind the marker purifying and reach more than 95%,
125I/
131Modify on each KH901 adenovirus in the I-KH901 modified outcome
125I/
131I is about 8.32～9.25 * 10
-3Bq.
The adenovirus of embodiment two preparation is placed on 4 ℃ of refrigerators, measures marker radiochemical purity of different time in 1,2,3,4,5,6,7 day behind mark with Paper Chromatography.The result shows
125I/
131It is basicly stable that I-KH901 places 7 days its radiochemicsl purities at 4 ℃ of refrigerators, remains on 93%～95%, shows that it has stronger stability external.In order in clinical, to use more convenient safety after this product is convenient to, this product can be stored under-80 ℃ of conditions.
Biodistribution and Detection of Stability in the adenovirus body that embodiment three radionuclides of the present invention are modified
At present, radionuclide is used for oncotherapy both can directly be used at tumor by local, also can treat by antibody target.With the viral oncolytic therapy and the radio nuclide therapy combination of tumour, utilize viral oncolytic and nucleic Synergistic killing, kill tumor cell for better; Simultaneously, for biology characteristics in the body of inquiring into KH901, the present invention carries out
125The intravital biodistribution experiment of I-KH901 normal mouse had both been studied
125The body internal stability of I-KH901 also is that the further research of the oncolytic therapy of KH901 lays the foundation
Prepare 8 groups of mouse, 5 every group, every mouse is 94% through tail vein injection 0.1ml radiochemicsl purity
125I-KH901.Other gets two 0.1ml
125I-KH901 is as standard source.Respectively take out one group of mouse broken end respectively at injection back 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h and get blood, dissect and take out each main organs, weigh and measure its radiocounting.The result represents with every gram tissue radiation picked-up per-cent (%ID/g).
125The I-KH901 marker is listed in table 1 in the intravital bio distribution of normal mouse.The result shows
125I-KH901 mainly is distributed in the liver, secondly content is also higher in kidney, spleen, the prolongation in time of the radioactive uptake of each internal organs constantly descends in the 24h except that liver and kidney, and the increased radioactivity of liver and kidney is still higher behind the 24h, and the increased radioactivity of enteron aisle is lower always
Use radionuclide
125I carries out the biodistribution test in the normal mouse body of mark and marker to specificity oncolytic recombinant adenovirus KH901, and the result shows: KH901 can by
125I modifies, and the purified back of modifier vitro stability is good, and putting is pure after 7 days remains on 93%～95% 4 ℃ of external placements.Marker mainly is distributed in liver in the normal mouse body, secondly content is also higher in kidney, spleen.The radioactive uptake prolongation in time of each internal organs constantly descends in the 24h, and the increased radioactivity of liver and kidney is still higher behind the 24h, and the increased radioactivity of enteron aisle is lower always.
The biodistribution experiment shows in the body
125I-KH901 absorbs higher in liver, and the residence time is longer, and the cue mark thing may be bigger to the toxic side effect of liver, and on the other hand, also the effect of marker to liver cancer cell can be further studied in prompting.Radioactivity is lower and remove comparatively fast in blood, and background may be lower when its video picture was described, the potential using value as developer is arranged.Radioactivity in the Tiroidina is higher, may be owing to do not seal Tiroidina, the picked-up free
125Due to the I.The increased radioactivity of each internal organs is along with the prolongation of time is totally on a declining curve, and the increased radioactivity of liver and kidney is still higher after 24 hours, and the increased radioactivity of enteron aisle is lower always.
The biologic activity of KH901 detects behind embodiment four radioisotope labelings
This experiment and result are as follows:
Because the cDNA of GM-CSF is assigned in the E3 district of KH901, can be at tumor locus expression of GM-CSF factor, the antitumor action of enhancing body.Shen Fubing etc. have confirmed by experiment that KH901 expresses high-caliber granulocyte-monocyte G CFS (GM-CSF) in tumour cell, and have only a small amount of GM-CSF to produce in normal cell.This experiment is by with MOI=10PPC and MOI=1 PPC
131I-KH901 infected person tumour cell adopts the ELISA method to measure the immunocompetence of GM-CSF after 24 hours.Can reflect indirectly
131The biologic activity of I-KH901 marker.
131The detection of I-KH901 expression of GM-CSF in tumor cell in vitro: HepG2 presses with human tumor cells
1 * 10
6/ hole is inoculated in 24 porocyte culture plates, when treating that cell covers with at the bottom of the hole, respectively with MOI=10 PPC and MOI=1PPC
131I-KH901 infects each porocyte of culture plate, then at 37 ℃, 5%CO
2Hatched respectively in the incubator 24 hours, difference collecting cell supernatant nutrient solution, (press the operation of test kit specification sheets, test kit is BPB Biomedicals, and Inc measures the GM-CSF immunocompetence with the ELISA method.
Statistical analysis: all experimental datas with mean ± standard deviation (x ± s) expression adopts SPSS 13.0 statistical softwares to analyze, the P value less than 0.05 for statistical significance is arranged.Relatively checking of two kinds of methods with designing t in groups.
The different infection multiplicities of table 2
131The expression of I-KH901 GM-CSF in tumor cell in vitro
| Cell | ?MOI | Incubation time | ?ELISA(pg/ml) |
| HepG2 | ?1 | ?24h | ?181.173±5.3418 |
| HepG2 | ?10 | ?24h | ?183.279±8.4385 |
The results are shown in table 2.As seen, MOI=10 PPC and MOI=1 PPC
131During I-KH901 infected person tumour cell HepG2, can express a large amount of GM-CSF, P＞0.05, no difference of science of statistics between the two.Show that radio-labeling handles the biological activity of adenovirus is not had influence.
Embodiment five the present invention
131I-KH901 is to the treatment experiment of the nude mice of lotus people liver cancer
The external specificity antineoplastic effect of adenovirus KH901 studies show that
[6]KH901 optionally duplicates in tumour cell, and replication is poor in normal cell.Shen Fubing
[1]Deng confirming that KH901 has antitumor action in the mice with tumor body, and express high-caliber GM-CSF.Above KH901 experiment in vivo and vitro shows that all it can become a kind of up-and-coming clinical gene therapy medicine.But KH901 biological behaviour in vivo is not clear, adopts general drug metabolism study method to understand.So the present invention
125I/
131Effect just is difficult to prediction more in the body of I-KH901.
HepG2 transplanted tumor in nude mice model construction: selectivity index phase grown cell counting reaches 10
6～10
7The HepG2 cell of cells/ml, the centrifugal 5min of 1000r/min, supernatant discarded, with PBS with cell resuspended be concentration 5 * 10
6～1 * 10
7The single cell suspension of cells/ml, standby.Get 20 nude mices, it is subcutaneous only fast single cell suspension to be inoculated in the inboard armpit of nude mice right fore by 0.2ml/.Aseptic principle is followed in the experimental procedure strictness, raises in West China medical experiment animal center.About 2 weeks, tumour is grown to about 0.8cm * 1.0cm, is used for experiment.
The treatment of liver cancer nude mice: experiment grouping: get 20 lotus people liver cancer nude mices, grade by body weight, tumour size is divided into 4 groups, 5 every group, again the tumor bearing nude mice in each group is assigned randomly to each experimental group and control group, every group 4, accept different treatment respectively.
A, negative control group (A group): every tumor bearing nude mice is through tail vein injection 0.2ml physiological saline;
B, positive controls (B group): every tumor bearing nude mice is through tail vein injection 0.1mg/0.2ml pidorubicin;
C,
131I-KH901 knurl body injection group (C group): every tumor bearing nude mice is directly slowly injected 0.2ml through the tumor center position
131I-KH901 (1.52MBq
131I, 1.6 * 10
9The every mouse of VP/);
D,
131I organizes (D group): every tumor bearing nude mice is through tail vein injection 0.2ml Na
131I (1.52MBq);
E, KH901 knurl body injection group (E group): every tumor bearing nude mice is directly slowly injected 0.2mlKH901 (1.6 * 10 through the tumor center position
9The every mouse of VP/)
Embodiment: each treated animal of accepting different treatment is all raised at experimentation on animals center, identical SPF level West China, and 3 d finish oral 0.1%KI solution with the protection Tiroidina to experiment before administration, positive controls, every 3 all repeat administrations once; A group, C group, D group and E group all at interval 2 days repeat administrations once, administration is 3 times altogether.
The tumor growth situation detects: experimental session is observed tumor growth situation (surface has or not ulcer, necrosis etc.) every 3d timing special messenger, and with the size (major diameter a, minor axis b) of vernier caliper measurement tumour, the tumour size is respectively organized in observation continuously, finishes until experiment, and the observation period is January.By formula calculate gross tumor volume and inhibition rate of tumor growth:
Gross tumor volume (cm
3) V=ab
2/ 2
Tumor control rate (%)=(1-(the treatment group begins heavily treatment group of average knurl and finishes average knurl heavily)/(control group begins the heavy control group of average knurl and finishes average knurl heavily)) * 100%
Statistical analysis: all experimental datas with mean ± standard deviation (x ± s) expression adopts the SPSS13.0 statistical software to analyze, the P value less than 0.05 for statistical significance is arranged.Two kinds of methods relatively use paired t-test, the variance analysis of relatively adopting the completely random design between each tumor bearing nude mice treatment group, each variable relatively adopts variance analysis between each group.
Experimental result shows that respectively organizing the tumor growth situation sees Table 3.
The variation of gross tumor volume before and after table 3 administration (n=4, x ± s)
As seen, each treatment group gross tumor volume no difference of science of statistics before the administration,
131I-KH901 passes through the intratumor injection administration, with
131The administration group compares in I tail intravenously administrable group and the KH901 knurl, and tumour inhibiting rate has significant difference;
131Administration group tumour inhibiting rate and positive controls relatively have significant difference in I group, the KH901 knurl, compare no difference of science of statistics with negative control group.As can be seen,
131I-KH901 has therapeutic action to lotus people liver cancer nude mice, and has shown in this experiment than using separately
131I group or the better effect of KH901.In addition, this experimental session, positive controls has a tumor bearing nude mice can not tolerate chemotherapeutics toxicity and death, and surplus tumor bearing nude mice all tolerates treatment, does not have dead.
131After the I-KH901 treatment, the tumor bearing nude mice generalized case is improved, and appetite increases, and weight increase is normal.
Embodiment six the present invention
131I-KH901 is to the video picture experiment of the nude mice of lotus people liver cancer
131I-KH901 is at the intravital radionuclide imaging of tumor bearing nude mice:
(1 of the nude mice of the about 0.8cm of tumour * 1.0cm), it is fixing to lie on the back, through intratumor injection 0.2ml to take out lotus HepG2 knurl
131I-KH901 (3.7MBq); Get lotus knurl (1 of the nude mice of the about 0.8cm of tumour * 1.0cm), the Na of injection same dose in the knurl body again
131I, in contrast.
Carry out static state respectively at 2h, 6h, 12h, 24h separately after the injection and gather radionuclide image, observe
131I-KH90 and Na
131I is in the dense poly-situation of tumor locus.
Tumor bearing nude mice video picture result: as shown in Figure 2, injection
131The tumor bearing nude mice of I-KH901 all has very strong retention of activity from start to finish in the knurl body, the radioactive uptake that prolongs tumor locus is not in time seen obviously and weakened, the still clear development of tumour behind the 24h, and position, size still conform to actual; And during whole video picture, only when 2h, 6h, have outside the bladder image, there is no other tissue has tangible increased radioactivity.But, injection Na
131The tumor bearing nude mice of I is all obviously seen bladder image (see figure 3) from start to finish, prolongs bladder in time, the neck video picture has the enhanced sign, and during whole video picture, the video picture of the tumor locus that only when 2h, mays be seen indistinctly, all the other times there is no tangible tumor imaging.This experiment shows that the adenovirus KH901 of radioisotope labeling of the present invention can be enriched in tumor locus and continue the performance function.
Above more excellent embodiment is that the present invention is further illustrated, but be not limitation of the scope of the invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope that spirit of the present invention and claims of being enclosed define.
Reference:
1, Shen Fubing, Yang Chun, Lei Ning etc.Specificity oncolytic recombinant adenovirus KH901 is to the restraining effect of mice with tumor tumor growth and the research of expression of GM-CSF.Sichuan University's journal (medicine), 2007; 38 (3): 286-390
2、Spear?MA，Sun?F，Eling?DJ，et?al.Cytotoxicity，apoptosis，and?viralreplication?in?tumor?cells?treated?with?oncolytic?ribonucleotidereductase-defective?herpes?simplex?type?1?virus(hrR3)combined?with?ionizingradiation.Cancer?Gene?Ther，2000；7：1051-1059
3、Tang?Dc，Jennelle?RS，Shi?Z，Carbone?DP，Loya?F，Chang?CH，et?al.Overexpression?of?adenovirus-encoded?transgenes?from?the?cytomegalovirusimmediate?early?promoter?in?irradiated?tumor?cells.Hum?Gene?Ther，1997；8(17)：2117-2124
4、Bradley?JD，Kataoka?Y，Advani?S，Chung?SM，Arani?RB，Gillespie?GY，et?al.Ionizing?radiation?improves?survival?in?mice?bearing?intracranial?high-gradegliomas?injected?with?genetically?modified?herpes?simplex?virus.Clin?CancerRes，1999；5：1517-522
5、Mather?SJ，Ward?BG..High?efficiency?iodination?of?monoclonal?antibodiesfor?radiotherapy.J?Nucl?Med，1987；28(6)：1034-1036
6, Shen Fubing, Chang Jianhua, Yang Chun, etc.The external specificity antineoplastic effect research of specificity oncolytic recombinant adenovirus KH901.Sichuan University's journal (medicine), 2007; 38 (1): 31-34
Claims (9)
1. the adenovirus of radioisotope labeling is characterized in that radionuclide is modified on the adenovirus capsid albumen, and the radionuclide of modifying on each adenovirus is 5.0 * 10
-3～2.0 * 10
-2Bq.
2. the adenovirus of radioisotope labeling according to claim 1 is characterized in that described radionuclide is
125I,
131I,
32P,
188Re,
186Re,
90At least a among the Y.
3. the adenovirus of radioisotope labeling according to claim 1 is characterized in that described adenovirus is the oncolytic adenovirus.
4. the adenovirus of radioisotope labeling according to claim 3 is characterized in that described oncolytic adenovirus is adenovirus KH901.
5. the adenovirus of radioisotope labeling according to claim 1 is characterized in that the radionuclide of modifying on described each adenovirus is 8.3 * 10
-3～9.3 * 10
-3Bq.
6. prepare the method for the adenovirus of each described radioisotope labeling of claim 1～5, it is characterized in that may further comprise the steps:
Adding concentration is 1 * 10
8～1 * 10
10The adenovirus solution of VP/mL adds 1 * 10 in per then 100 μ l adenovirus solution
5～1 * 10
7The radionuclide of Bq, per again 100 μ l add N-bromosuccinimide (NBS) 1～100 μ g, add phosphate buffered saline buffer under the room temperature and regulate pH value to 7～7.6, react 2～5 minutes, add concentration again and be human serum albumin solution's termination reaction of 2%.
7. the method for the adenovirus of preparation radioisotope labeling according to claim 6 is characterized in that may further comprise the steps:
Preparing concentration is 8 * 10
9The adenovirus KH901 of VP/mL, per then 100 μ l adenovirus solution add 7.4MBq
131I/
125I and 25 μ g NBS add the pH value and are the phosphate buffer 1 20 μ l of 7.5 0.05mol/L again, react 3min under the room temperature, add human serum albumin solution's termination reaction of 10 μ l 2% again.
8. the adenovirus of each described radioisotope labeling of claim 1～5 is in the purposes of preparation in the antineoplastic pharmaceutical compositions.
9. antineoplastic pharmaceutical compositions is that main active ingredient is added pharmaceutically acceptable complementary composition and is prepared from by the adenovirus of each described radioisotope labeling of claim 1～5.
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