CN1318095C - Tracer agent for oxygen lacking tumor cell and its usage - Google Patents
Tracer agent for oxygen lacking tumor cell and its usage Download PDFInfo
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- CN1318095C CN1318095C CNB2003101089048A CN200310108904A CN1318095C CN 1318095 C CN1318095 C CN 1318095C CN B2003101089048 A CNB2003101089048 A CN B2003101089048A CN 200310108904 A CN200310108904 A CN 200310108904A CN 1318095 C CN1318095 C CN 1318095C
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Abstract
The present invention belongs to a nuclear medicine technical field which relates to a radionuclide anoxia and tumor visualization tracer, and particularly to a radionuclide visualization tracer deacidizing free 99<Tc><m> and 188<Re> for tumors and ischemia and hypoxia diseases. The present invention makes use of an organic solution as an agent for the disproportionating reaction, and reducing agents are used to deacidize high 99<Tc><m> acid salt and high 188<Re> acid salt into stable low valence free 99<Tc><m> and free 188<Re>. The experiment confirms that the body distributing index of the deacidized free 99<Tc><m> is obviously superior to the distributing index of other anoxic imaging agents, the tumour visualization has ideal effect, and toxicity and a killing function in anoxia tumor cells are obviously increased. As a quite ideal anoxia and tumor tracer, the present invention can be used in diagnoses and treatment of tumour radioactivity visualization, and diagnoses of ischemia and hypoxia diseases and ischemic heart diseases.
Description
Technical field
The invention belongs to the field of nuclear medicine, be specifically related to weary oxygen of a kind of active nucleus and tumor imaging tracer and the purposes in the nucleic of tumor and weary oxygen disease radiates localization diagnosis and treats thereof.
Background technology
In the clinical diagnosis to tumor, the diagnosis of nucleic irradiation image has extraordinary proper value.Wherein the most desirable and clinical extensive use be fluoro-18 fluorodeoxyglucose Positron Emission Computed Tomographies (
18F-FDG PET), yet,
18Still there is following shortcoming in F-FDG PET: 1,
18F-FDG PET checks and costs an arm and a leg that once checking needs the cost RMB more than 10,000 yuan; 2, the positive imaging results of the pathological changes of the normal structure organ (as brain, liver etc.) of hypermetabolism is poor; 3, the high energy positron nucleic
18F does not have therapeutic value for tumor.Compare with PET, the inspection cost of single photon emission computed tomography (SPECT) is much lower relatively.Though some single photon nucleic chemical compound also has close tumor feature, does not up to the present still have to substitute positron
18The single photon nucleic chemical compound of F-FDG.Single photon nucleic technetium-99m (
99Tc
m) not only low price, and convenient sources, can the labelling chemical compound lot, be global most widely used nucleic at present.
99Tc
mLabelling parent's tumor chemical compound such as monoclonal antibody, polypeptide, antitumoral compounds, energy metabolism chemical compound and weary oxygen compound etc. have all been obtained some progress, but still are difficult to substitute positron
18F-FDG.Wherein,
99Tc
mThe weary oxygen compound of labelling has extraordinary application prospect, and this is not only because there is anoxia state in most of entity tumor, and it is all insensitive to radiotherapy and chemotherapy to be in the tumor cell of anoxia state, is one of oncotherapy very thorny problem.Therefore,
99Tc
mThe weary oxygen compound of labelling not only can be used for diagnosing tumour, utilize it the Auger electron radiation feature or its analog beta-particle emission nucleic rhenium-188 (
188Re) can be used for oncotherapy.
Existing anoxia developing agent preferably comprises at present
18F-FMISO,
123I-IAGA,
99Tc
m-Nitroimi-dazole (BMS) and
99Tc
m-HL91 is wherein with non-nitro glyoxaline
99Tc
m-HL91 is the most desirable, nonetheless,
99Tc
m-HL91 still is difficult to be extensive use of clinical.
Summary of the invention
The purpose of this invention is to provide a kind of weary oxygen and neoplasm tracing agent that is used for tumor and hypoxic-ischemic disease radioactivity localization diagnosis, be specifically related to the nucleic reduction and dissociate
99Tc
mFree with reduction
188Re is as weary oxygen and neoplasm tracing agent.It is free that further purpose of the present invention provides reduction
99Tc
mFree with reduction
188The purposes of Re treatment tumor.
The present invention as acetonitrile, as the dismutation reaction media, adopts Reducing agent, as stannous chloride, with height with organic solution
99Tc
mHydrochlorate (sodium) and height
188It is free that Re hydrochlorate (sodium) is reduced into stable low price
99Tc
mWith free
188Re, and through experiment confirm, reduction is free
99Tc
mFree with reduction
188Re is a kind of very ideal weary oxygen and neoplasm tracing agent, can be used for tumour radiotherapy localization diagnosis and treatment, and the diagnosis of other hypoxic-ischemic diseases (as ischemic heart desease).
Organic solvent of the present invention is to dewater and immiscible as the organic solvent beyond ethyl acetate, the chloroform etc. with water, and as acetonitrile, acetone etc., described Reducing agent is stannous chloride, sodium thiosulfate, ferrous sulfate, sodium borohydride etc.
The present invention realizes by following method
One, the preparation reduction is free
99Tc
mFree with reduction
188Re
With the organic solvent is media, adopts Reducing agent (as stannous chloride) with height
99Tc
mHydrochlorate (sodium) and height
188It is free that Re hydrochlorate (sodium) is reduced into stable low price
99Tc
mWith free
188Re.
High
99Tc
mHydrochlorate (
99Tc
mO
4 -) and high
188The Re hydrochlorate (
188ReO
4 -) at Reducing agent, be reduced into stable down earlier as effects such as stannous chloride, sodium thiosulfate, ferrous sulfate
99Tc
mColloid and
188The Re colloid, the latter passes through organic solvent again, and as effects such as acetonitrile, acetone, it is free to form two kinds of stable reduction through dismutation reaction
99Tc
mFree with reduction
188Re.
Two, height
99Tc
mAcid (
99Tc
mO
4 -) reduction reaction and
99Tc
mColloidal dismutation reaction
1, reduction reaction: in aseptic Dewar bottle, add 0.1ml's
99Tc
mO
4 -After (about 148MBq), add the reducing solution 15 μ l that contain the 150ug stannous chloride, reduction reaction 5min under the room temperature.Adopt ply of paper to analyse with the silica gel plate chromatography and analyze, developing solvent is respectively water and acetone.During ply of paper is analysed
99Tc
mO
4 -With
99Tc
mColloidal Rf value is respectively 0.70-0.75 and 0.00-0.10; The silica gel plate chromatography
99Tc
mO
4 -With
99Tc
mColloidal Rf value is respectively 0.97-1.00 and 0.00-0.10.The result shows, 100%
99Tc
mO
4 -Be reduced into
99Tc
mColloid.
2, dismutation reaction: in aseptic Dewar bottle, add 0.1ml's
99Tc
mBehind the colloid, add the water of 0.4ml-0.6ml or the organic solvent of 0.4m1-0.6ml again, react 25min under the room temperature.Adopt ply of paper to analyse and the silica gel plate chromatography, developing solvent is respectively water and acetone.Two kinds of reduction were free during ply of paper was analysed
99Tc
mRf value be respectively 0.80-0.85 and 0.70-0.75,
99Tc
mColloidal Rf value is 0.00-0.10; Two kinds of reduction are free in the silica gel plate chromatography
99Tc
mRf value be respectively 0.95-1.00 and 0.80-0.85,
99Tc
mColloidal Rf value is 0.00-0.10.The result shows, organic solvent to the disproportionation ability of colloid technetium from by force to weak acetone 96.61%, acetonitrile 94.00%, oxolane 90.20%, dichloromethane 86.49%, acetic acid 84.83%, ethanol 68.58%, normal hexane 67.95%, toluene 59.68%, the N of being respectively, dinethylformamide 57.83%, ethyl acetate 47.22%, chloroform 44.15%, methanol 42.71%, ether 37.78% and water 0.00%.In tested organic solvent, wherein acetone, acetonitrile and oxolane make more than 90%
99Tc
mThe colloid disproportionation is free for reduction
99Tc
m, and water is right
99Tc
mColloid does not have disproportionation.
Three, select the preparation reduction free
99Tc
mOrganic solvent
Learn that by above-mentioned dismutation reaction test acetone, acetonitrile and oxolane are right
99Tc
mColloidal disproportionation is the strongest, and this can not determine that still acetone, acetonitrile and oxolane are exactly that best preparation stability reduction dissociates
99Tc
mOrganic solvent because Reducing agent exists all the time in practical operation, Reducing agent still needs to consider to the influence of dismutation reaction.Therefore, the present invention continues reduction-disproportionation-restore experiment, reduction-disproportionation-restore experiment: in aseptic vacuum, add 0.4m1-0.6ml water or following organic solvent earlier, after adding the reducing solution 15 μ l of the freshly prepared 150ug of containing stannous chloride again, add 0.1ml's
99Tc
mO
4 -(about 148MBq) reacts 15min under the room temperature, adds the water 0.2ml that contains the inferior stannum of excess chlorination (5mg/ml) at last again.The result shows, the disproportionation ability from strong be respectively acetonitrile 91.04%, N to weak organic solvent, dinethylformamide 54.89%, acetone 51.01%, acetic acid 38.03%, methanol 34.47%, oxolane 33.61%, dichloromethane 31.77%, toluene 25.87%, ether 23.38%, normal hexane 15.33%, chloroform 13.91%, ethanol 12.19%, ethyl acetate 7.06% and water 0.00%.
In tested organic solvent, wherein acetonitrile makes more than 90%
99Tc
mThe colloid disproportionation is free for reduction
99Tc
m, be the best reduction organic solvent.The disproportionation of DMF and acetone is about 50%, and the disproportionation of other organic solvents is below 40%, and water is right
99Tc
mColloid does not have disproportionation.
Four, reduction is free
99Tc
mFree with reduction
188Re
Method 1: in aseptic Dewar bottle, according to
99Tc
mO
4 -Or
188ReO
4 -The actual input amount of leacheate adds the strong organic solvent of an amount of disproportionation, as acetonitrile, and organic solvent volume: leacheate volume>3: 1.Add the freshly prepared Reducing agent of 50ug-250ug,, add fresh then at once as stannous chloride
99Tc
mO
4 -Or
188ReO
4 -Leacheate, reaction is at least 15 minutes under the room temperature.Utilize negative inferior pump, organic solvent in the aseptic Dewar bottle and the whole evaporations of water are drained, occur till the micro-pale solid thing to the bottle end.Staying water that the white solid thing is thoroughly dissolved the back with aseptic steaming uses.The radiation productive rate that obtains is greater than 85%.
Method 2: in aseptic Dewar bottle, add the freshly prepared Reducing agent of 50ug-250ug successively, as stannous chloride and fresh
99Tc
mO
4 -Or
188ReO
4 -Leacheate adds an amount of organic solvent then, as acetonitrile, and organic solvent volume: leacheate volume>3: 1.Reaction is at least 15 minutes under the room temperature.Utilize negative inferior pump, organic solvent in the aseptic Dewar bottle and the whole evaporations of water are drained, occur till the micro-pale solid thing to the bottle end.Stay water that the white solid thing is thoroughly used after the dissolving as early as possible with aseptic steaming.The radiation productive rate that obtains is greater than 85%.
Five, free
99Tc
mO
4 -Free with reduction
99Tc
mPhysics, chemistry and biology characteristic relatively 1, the comparison of physical characteristic: adopt thin layer chromatography (TLC), paper electrophoresis and oppositely degree of passing high performance liquid chromatography (HPLC) (HPLC) all to offer an explanation two kinds of reduction free
99Tc
m, wherein a kind of 80%-90% that accounts for is with free
99Tc
mO
4 -Can not distinguish mutually.Reduction is free
99Tc
mWith free
99Tc
mO
4 -From negative electrode anode electrophoresis, voltage is 300V, and electrophoresis time is 30 minutes, and electrolyte is PBS (PH=7.4).Reduction is free
99Tc
m(left side) and free
99Tc
mO
4 -Creep on (right side) all anode, illustrates that reduction is free
99Tc
mWith free
99Tc
mO
4 -Be anion.Wherein, reduction is free
99Tc
mHave two kinds, a kind of 80%-90% that accounts for is with free
99Tc
mO
4 -Can not distinguish mutually, the about 3cm of the distance of creeping, another kind of and free
99Tc
mO
4 -Difference accounts for 10%-20%, the about 6cm of the distance of creeping.2, the comparison of chemical characteristic: will dissociate
99Tc
mO
4 -Free with reduction
99Tc
mAdd compounds containing thiol groups respectively, in the solution as 3-mercaptoethanol, labeled reactant is 15 minutes under the room temperature, and labeled reactant condition 1 is PBS; Labeled reactant condition 2 is a 25mM lactic acid; Labeled reactant condition 3 is a 65mM lactic acid; Labeled reactant condition 4 is 1mM CaCl
2+ 6mM NaCl; Labeled reactant condition 5 is 6mM CaCl
2+ 6mM NaCl; Labeled reactant condition 6 is 65mM lactic acid+6mM CaCl
2+ 6mM NaCl.The result shows that under aerobic and weary oxygen condition, lactic acid can not promote reduction free
99Tc
mCombine with two mercapto propane sulfonic acid sodium, calcium ion can promote reduction free under anoxia state
99Tc
mCombine with two mercapto propane sulfonic acid sodium, it is many to make combination rate increase twice, and combination rate is apparently higher than aerobic state.The result shows, and is free
99Tc
mO
4 -Can not any compounds containing thiol groups of labelling, and reduction is free
99Tc
mCan the labelling compounds containing thiol groups, mark rate 10%-70%.Illustrate that reduction is free
99Tc
mChemical property active.Confirm that clinical usefulness contains the sulfydryl antidote, and to suppress in-vivo tumour picked-up reduction free
99Tc
m
3, the comparison of biological characteristics: (1) injects free respectively from nude mice tail vein
99Tc
mO
4 -Free with reduction
99Tc
m, injection back 1,2 and 4 hour, distribution character difference in the body of two kinds of chemical compounds, both are significant difference on liver, lung, spleen, pancreas, kidney, stomach, colon and thyroid distribute.(2) reduction is free
99Tc
mAnoxic cell or tissue there are affinity interaction, and dissociate
99Tc
mO
4 -Anoxic cell or tissue there is not affinity interaction.Table 1 is the interior distribution of the body of two kinds of chemical compounds, and table 2 is that reduction is free
99Tc
mWith free
99Tc
mO
4 -Anoxic cell or tissue there is affinity interaction.
Six, reduction is free
99Tc
mTo the affinity interaction of anoxic cell or tissue with free
99Tc
mO
4 -Comparison
The cell or tissue hypoxic-ischemic can cause a series of Developmental and Metabolic Disorder, because reduction is free
99Tc
mIon characteristic, it is free to reducing that the Developmental and Metabolic Disorder that following hypoxic-ischemic causes may change hypoxic-ischemic cell (or tissue)
99Tc
mPicked-up.Described abnormal metabolism comprises that the cell of local organization lacks oxygen, osmotic pressure increase, pH value decline, the accumulation of extracellular potassium ion, intracellular calcium overload, oxygen-derived free radicals generation etc.Result of the present invention confirms that weary oxygen, pH value descend, the extracellular potassium ion is accumulated and the intracellular calcium overload can promote that all H460 lung carcinoma cell picked-up reduction is free
99Tc
m, weary oxygen, pH value decline then suppress the picked-up of H460 lung carcinoma cell on the contrary and dissociate
99Tc
mO
4 -Following at normal, the accumulation of extracellular potassium ion, weary oxygen and pH value decline state, the H460 lung carcinoma cell is free to reducing
99Tc
mIntake be respectively
99Tc
mO
4 -11.9,13.3,191.0 and 63.7 times of cellular uptake amount, wherein the weary oxygen of histiocyte is that the reduction of decision hypoxic-ischemic cellular uptake is free
99Tc
mGreatest factor.In addition, cell is under anoxia state, and pH value descends and the calcium ion overload is to influence the cellular uptake reduction to dissociate
99Tc
mKey factor.
Experiment confirm of the present invention, reduction is free
99Tc
mEvery body in distribution index obviously be better than other anoxia developing agents, tumor imaging is satisfactory for result.Reduction is free
99Tc
mIn the tumor subcellular fraction distributes, have 40%-65% to be distributed in the nucleus, it is free obviously to increase reduction
99Tc
mTo the toxicity and the lethal effect of hypoxic tumor cells, in addition, free
188Re is the therapeutic nucleic of emission beta-particle, and reach 17 hours its physical half time, and after the intravenous injection 17 hours, radioactivity has mainly concentrated in the hypoxic tumor, and therefore, reduction is free
188Re treatment tumor efficiency is better.Reduction is free
99Tc
mAlso can be to ischemic diseases, as coronary heart disease, diagnose and assess.
Table 1 is free
99Tc
mO
4 -Free with reduction
99Tc
m, distribute in 1,2 and 4 hour the body in injection back.
Table 2 is that reduction is free
99Tc
mWith free
99Tc
mO
4 -The % cell binding ratio of hypoxic-ischemic under the different metabolic state.
Table 3 is that reduction is free
99Tc
mWith distribute relatively in the body of known anoxia developing agent.
Table 4 is that reduction is free
99Tc
mAfter the injection, distribute relatively in the body at nude mice normal tissue cell nuclear and lotus neoplastic cell nuclei.
Table 1
Injected back 1 hour | Injected back 2 hours | Injected back 4 hours | ||||||
%ID/g | Free technetium | The free technetium of reduction | %lD/g | Free technetium | The free technetium of reduction | %ID/g | Free technetium | The free technetium of reduction |
Blood | 4.826 | 5.481 | Blood | 3.121 | 3.369 | Blood | 2.435 | 2.449 |
Heart | 1.562 | 1.957 | Heart | 1.468 | 1.082 | Heart | 0.809 | 0.966 |
Liver | 3.678 | 9.323 | Liver | 2.413 | 6.758 | Liver | 2.500 | 6.641 |
Lung | 3.162 | 11.953 | Lung | 1.954 | 4.971 | Lung | 1.230 | 4.22 |
Thymus | 1.636 | 3.133 | Thymus | 2.505 | 1.291 | Thymus | 1.073 | 1.245 |
Spleen | 2.420 | 4.455 | Spleen | 0.795 | 3.849 | Spleen | 1.006 | 4.016 |
Pancreas | 5.011 | 1.764 | Pancreas | 2.164 | 1.205 | Pancreas | 1.903 | 0.756 |
Kidney | 2.883 | 7.720 | Kidney | 1.976 | 7.171 | Kidney | 1.700 | 7.409 |
The adrenal gland | 10.367 | 12.011 | The adrenal gland | 11.575 | 9.419 | The adrenal gland | 3.315 | 8.492 |
Gastric antrum portion | 14.65 | 16.368 | Gastric antrum portion | 24.48 | 14.63 | Gastric antrum portion | 12.24 | 7.155 |
Body of stomach | 6.820 | 9.689 | Body of stomach | 14.52 | 6.761 | Body of stomach | 17.13 | 6.862 |
Duodenum | 3.276 | 5.183 | Duodenum | 5.319 | 2.589 | Duodenum | 2.770 | 1.992 |
Small intestinal | 3.137 | 3.562 | Small intestinal | 3.624 | 3.304 | Small intestinal | 2.317 | 2.270 |
Colon | 2.064 | 1.179 | Colon | 7.291 | 1.007 | Colon | 1.717 | 0.299 |
Skeletal muscle | 1.728 | 1.591 | Skeletal muscle | 0.598 | 0.68501 | Skeletal muscle | 0.595 | 0.651 |
Thyroid | 72.01 | 92.518 | Thyroid | 123.5 | 47.71 | Thyroid | 82.27 | 21.63 |
Table 2
Different hypoxic-ischemic conditions | A | B | C | D | E | F | G |
Free 99Tc mO 4 -Reduction is free 99Tc m | 0.104 1.239 | 0.071 13.561 | 0.085 1.308 | 0.075 4.779 | 0.152 1.866 | 0.175 0.955 | 0.152 2.018 |
Table 3
Tumor | Tumor/skeletal muscle | Tumor/cardiac muscle | Tumor/lung | Tumor/liver | Tumor/spleen | Tumor/kidney | |
18F-FMISO 123I-IAGA 99Tc m-BMS 99Tc m-HL91 1 reduction is free 99Tc m2 reduction are free 99Tc m | 0.41 2.80 0.31 2.63 3.51 2.67 | 2.20 3.70 3.54 3.36 38.09 9.94 | 2.24 ---- 1.77 3.18 5.53 2.38 | 1.65 ---- 1.83 1.45 7.29 2.92 | 0.22 ---- 0.19 0.51 1.71 1.11 | ---- ---- ---- 1.24 5.01 2.01 | 0.51 ---- 0.56 0.68 2.39 0.92 |
Wherein, 1 reduction is free
99Tc
mBe human liver cancer HCCLM3-P15,
2 reduction are free
99Tc
mBe human H460 adenocarcinoma of lung.
Table 4
Injection back 2-4 hour | Injection back 18-20 hour | |
Heart liver kidney HCCLM3-P15 hepatocarcinoma H460 adenocarcinoma of lung | 18.56% 9.97% 12.05% 43.89% 40.44% | 18.94% 12.98% 13.87% 48.61% 63.10% |
Description of drawings
Fig. 1, the preparation reduction is free
99Tc
mFree with reduction
188The technology path figure of Re
Fig. 2, metabolic alterations is free to H460 lung adenocarcinoma cell picked-up reduction behind weary oxygen and the simulation ischemia
99Tc
mInfluence.
Fig. 3, lactic acid is free to the H460 lung adenocarcinoma cell picked-up reduction of aerobic and weary oxygen
99Tc
mWith free
99Tc
mO
4 -The comparison of influence.
Fig. 4, lactic acid, potassium, calcium and concentration of glucose are free to weary oxygen H460 lung adenocarcinoma cell picked-up reduction
99Tc
mInfluence.
Fig. 5, a routine lotus people H460 adenocarcinoma of lung nude mice tail vein injection reduction is free
99Tc
mAfter the video picture result.
Fig. 6, a routine lotus people HCC-M3-P15 hepatocarcinoma nude mice tail vein injection reduction is free
99Tc
mAfter the video picture result.
Fig. 7, the SD rat heart model perfusion result that exsomatizes,
Wherein, arrow indication place, left side is SD rat heart left ventricle lower wall ischemia place, and visible significantly reduction is free
99Tc
mRadiate dense poly-kitchen range, the right side
201Tl image same area shows no obvious abnormalities.
Fig. 8, the of short duration ischemia figure of new zealand rabbit left circumflex artery areal area cardiac muscle,
Wherein, right part of flg arrow indication place is
201The Tl pictorial display should the zone radiation defect on a large scale, reduction is free
99Tc
mInjection is early stage, the sensitive myocardial ischemia that shows.
Fig. 9, reduction is free
99Tc
mWith free
99Tc
mO
4 -The paper electrophoresis analysis relatively.
Figure 10, reduction is free
99Tc
mThe comparison of labelling two mercapto propane sulfonic acid sodium under aerobic and anoxia state.
The specific embodiment
Embodiment 1 external aerobic and anoxic cell picked-up reduction are free
99Tc
mWith free
99Tc
mO
4 -The human adenocarcinoma of lung H460 cell strain (providing) of comparison by U.S. The University of Texas MD AndersonCancer Center, be incubated in 24 well culture plates with RPMI-1640, the H460 cell is in the growing multiplication phase, reduces free in following different experimental conditions
99Tc
mWith free
99Tc
mO
4 -Cell in vitro in conjunction with experiment.The result shows at normal, the accumulation of extracellular potassium ion, weary oxygen and pH value decline state following, and the H460 lung carcinoma cell is free to reducing
99Tc
mIntake be respectively
99Tc
mO
4 -11.9,13.3,191.0 and 63.7 times of cellular uptake amount, wherein the weary oxygen of histiocyte is that the reduction of decision hypoxic-ischemic cellular uptake is free
99Tc
mGreatest factor.In addition, cell is under anoxia state, and pH value descends and the calcium ion overload is to influence anoxic cell picked-up reduction to dissociate
99Tc
mKey factor.Experiment condition is as follows:
Condition A: renew bright RPMI-1640 0.5ml, add reduction and dissociate
99Tc
mWith free
99Tc
mO
4 -After, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 1.24% ± 0.26% and 0.11% ± 0.02%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -11.9 times of cellular uptake amount.
Condition B: renew bright RPMI-1640 0.5ml, add reduction and dissociate
99Tc
mWith free
99Tc
mO
4 -After, take out negative pressure after 15 minutes, make the H460 cell lack oxygen, 37 ℃ of cell association reactions 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 13.56% ± 0.15% and 0.07% ± 0.002%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -191.0 times of cellular uptake amount.
Condition C: change and contain the fresh RPMI-1640 of 350mM sucrose (osmotic pressure 380mOsm), it is free to add reduction
99Tc
mWith free
99Tc
mO
1 -After, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 1.31% ± 0.19% and 0.07% ± 0.006%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -18.7 times of cellular uptake amount.
Condition D: use the fresh RPMI-1640 of 55mM succinic acid or lactic acid (PH=4.5) instead, add reduction and dissociate
99Tc
mWith free
99Tc
mO
4 -After, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 4.78% ± 0.47% and 0.07% ± 0.009%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -63.7 times of cellular uptake amount.
Condition E: change and contain the fresh RPMI-1640 of 0.5mM CaCl2+9mM NaCl, it is free to add reduction
99Tc
mWith free
99Tc
mO
4 -After, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 1.87% ± 0.48% and 0.10% ± 0.05%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -18.7 times of cellular uptake amount.
Condition F: change the fresh serum deprivation RPMI-1640 that contains 0.15% hydrogen peroxide+20 microgram copper sulfate, add reduction and dissociate
99Tc
mWith free
99Tc
mO
4 -After, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 0.96% ± 0.17% and 0.18% ± 0.06%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -5.4 times of cellular uptake amount.
Condition G: change and contain the fresh RPMI-1640 of 10mM KCl to add reduction free
99Tc
mWith free
99Tc
mO
4 -After, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, reduction is free
99Tc
mWith free
99Tc
mO
4 -% cellular uptake rate be respectively 2.02% ± 0.08% and 0.15% ± 0.01%, reduction is free
99Tc
mH460 lung carcinoma cell intake be
99Tc
mO
4 -13.3 times of cellular uptake amount.
Human adenocarcinoma of lung H460 cell strain (providing by U.S. The University of Texas MD AndersonCancer Center) is provided, cultivate in sub 24 well culture plates with RPMI-1640, the H460 cell is in the growing multiplication phase, takes out and causes cell to lack oxygen in negative pressure 20-30 minute.The result shows that under the aerobic situation, along with pH value descends, cellular uptake is free
99Tc
mO
4 -Progressively lower, on the contrary, when pH value dropped to 5.5, aerobic cell picked-up reduction was free
99Tc
mBeginning descends along with pH value and increases.Under weary oxygen situation, the cellular uptake reduction is free
99Tc
mPH value is changed sensitivity more, and when pH value dropped to 7.0, anoxic cell picked-up reduction was free
99Tc
mPromptly begin to descend and increase along with pH value, it is free to reducing that the pH value that local microenvironment be described changes energy appreciable impact anoxic cell
99Tc
mPicked-up, anoxic cell anerobic glycolysis metabolism is strengthened and the local microenvironment pH value that causes thus lowers and helps anoxic cell picked-up reduction free
99Tc
m
Cell hypoxia all can be followed intracellular calcium overload and the accumulation of extracellular potassium, this experiment confirm, and in the presence of calcium, anoxic cell picked-up reduction is free
99Tc
mObviously increase, illustrate that the calcium overload is to cause anoxic cell picked-up reduction free
99Tc
mAnother key factor.The concentration of potassium and glucose increases and promotes that anoxic cell picked-up reduction is free
99Tc
mEffect not obvious, do not have specificity yet.
Embodiment 3 anoxic cells picked-up reduction is free
99Tc
mMechanism experiment
Because reduction is free
99Tc
mAnion characteristic, it is free that cell membrane anion transport body (aniontransporter) may participate in reduction
99Tc
mTranshipment because under normal circumstances the anion transport body is in inactive state, but when pH value descended, cell membrane anion transport physical ability was activated, thereby promoted reduction free
99Tc
mEnter anoxic cell.
With no chemically active free
99Tc
mO
4 -Difference, reduction is free
99Tc
mHave chemism, can combine with the macromolecular compound that contains sulfydryl, amino, thereby residue in the cell.The following experiment of the present invention proves that weary oxygen can promote reduction free
99Tc
mCombine with the macromolecular compound that contains sulfydryl, amino.
Two mercapto propane sulfonic acid sodium contain two sulfydryls, and energy and melts combine are the metal poisoning antidotes of clinical use.Because reduction is free
99Tc
mHave chemism, also can combine with two mercapto propane sulfonic acid sodium.Under aerobic and weary oxygen condition, lactic acid can not promote reduction free
99Tc
mCombine with two mercapto propane sulfonic acid sodium, illustrate that sour environment promotes that the cellular uptake reduction is free
99Tc
mFree with reduction
99Tc
mLabelling contains sulfydryl, amino macromolecular compound does not have direct relation.Yet calcium ion can promote reduction free under anoxia state
99Tc
mCombine with two mercapto propane sulfonic acid sodium, it is many to make combination rate increase twice, and combination rate is apparently higher than aerobic state.Illustrate that calcium ion promotes that anoxic cell picked-up reduction is free
99Tc
mFree with reduction
99Tc
mLabelling contains sulfydryl, amino macromolecular compound ability strengthens having certain relation.
The result confirms that the sour environment that anoxic cell produces helps reduction free
99Tc
mEnter cell, and the intracellular calcium overload that weary oxygen causes helps reduction free
99Tc
mCombine with the macromolecular compound that contains sulfydryl, amino.
Cell membrane potential changes and cell membrane integrity pair cell picked-up reduction dissociates
99Tc
mInfluence
Physiology and pathological cells transmembrane potential change, and necrocytosis that perhaps weary oxygen or ischemia cause or cell membrane destroy whether pair cell picked-up reduction is free
99Tc
mExert an influence, free to determining reduction
99Tc
mWhether be that weary oxygen tracer is very important.Human adenocarcinoma of lung H460 cell strain (providing by U.S. The University of Texas MDAnderson Cancer Center) is provided, be incubated in 24 well culture plates with RPMI-1640, the H460 cell is in the growing multiplication phase, uses cell membrane depolarization liquid (containing 10mM KCl), cell membrane hyperpolarization liquid (containing the 1mM sodium tetraphenylborate) and cell membrane to destroy liquid (containing 0.5%Triton X-100) then and replaces the RPMI1640 culture fluid.It is free to add reduction
99Tc
mAfter, in CO2 gas incubator with H460 cell association reaction 3 hours.As a result, the reduction after normal control, cell membrane depolarization, cell membrane hyperpolarization and cell membrane destroy is free
99Tc
mThe cell combination rate is respectively 0.807% ± 0.062%, 1.614% ± 0.249%, 2.817% ± 1.608% and 0.272% ± 0.019%.Wherein, cell membrane destroys and makes the cellular uptake reduction free
99Tc
mObviously lower, illustrate that non-viable non-apoptotic cell does not absorb reduction and dissociates
99Tc
mNo matter cell membrane depolarization or hyperpolarization can both make the cellular uptake reduction free
99Tc
mIncrease to some extent.
Embodiment 5 reduction are free
99Tc
mWith free
99Tc
mO
4 -In normal nude mouse, distribute relatively
Nude mice (23-27 restrains body weight) fasting 6-12 hour, under waking state, the tail vein injection reduction is free
99Tc
mOr it is free
99Tc
mO
4 -About 15 μ Ci (555Bq), volume injected is less than 0.2ml.Put to death nude mice in 1,2 and 4 hour respectively at the injection back, get blood and each histoorgan and measure every gram tissue counts and injection grand total, calculate every gram tissue counts and account for injection grand total percentage ratio (%ID/g), the result shows that reduction is free
99Tc
mIn the increased radioactivity of liver, lung, spleen and kidney apparently higher than free
99Tc
mO
4 -On the contrary, injected back 2 hours, reduction is free
99Tc
mBe starkly lower than free in pancreas, stomach, colon and thyroid increased radioactivity
99Tc
mO
4 -Results suggest, reduction is free
99Tc
mEasily absorbed, and dissociate by the normal structure organ
99Tc
mO
4 -Be more prone to drain from digestive tract.The present embodiment result is consistent in conjunction with experimental result with cell in vitro, and aerobic cell is free to reducing
99Tc
mIntake is free
99Tc
mO
4 -11.9 times.
Human liver cancer HCCLM3-P15 cell strain (purchasing mountain hospital liver cancer research institute in Fudan University) and human H460 lung adenocarcinoma cell line (being provided by U.S. The University of Texas MD Anderson CancerCenter) are incubated in the RPMI1640 culture fluid (SIGMA product) that contains calf serum.To breed the right side oxter of phase tumor cell inoculation in nude mice (purchasing) in the Shanghai Inst. of Tumor.In inoculation 4 week-10 weeks of back, tumor is grown up to using more than the 0.5cm.Tumor bearing nude mice is under waking state, and the tail vein injection reduction is free
99Tc
mAbout 250 μ Ci (9250Bq), volume injected is less than 0.2ml.The anesthesia in 2,4,8,12 and 18 hours of injection back, put to death tumor bearing nude mice after the video picture, get blood, tumor and each histoorgan and measure every gram tissue counts and injection grand total, calculate every gram tissue counts and account for injection grand total percentage ratio (%ID/g), calculate the %ID/g ratio of tumor and normal structure at last.The result shows that reduction is free
99Tc
mTumor and the %ID/g ratio of blood, skeletal muscle, the heart, lung, liver,spleen,kidney all obviously be better than other anoxia developing agents, comprise best anoxia developing agent at present
99Tc
m-HL91.The result confirms that reduction is free
99Tc
mHaving remarkable close hypoxic tumor characteristic, is a breakthrough of hypoxia imaging research, and reduction is free
99Tc
mTo become optimal anoxia developing agent.The reduction of lotus people H460 adenocarcinoma of lung nude mice tail vein injection is free
99Tc
m2,6 and 18 hours the video picture in back is injected back 2 hours video picture tumors and is got final product clear demonstration (there is necrosis region in central authorities), and 18 hours, main radioactive dosage all concentrated in the tumor tissues, has only residual a little radioactivity of liver stomach function regulating.The reduction of lotus people HCC-M3-P15 hepatocarcinoma nude mice tail vein injection is free
99Tc
m4,8 and 20 hours the video picture in back, about 0.7 * 0.7 * 0.4cm of tumor, the clear demonstration of tumor, thyroid, harmonization of the stomach liver also have than hot and distribute.
Embodiment 7 reduction are free
99Tc
mSubcellular fraction in tumor and normal structure distributes
99Tc
mBe the Auger electron active nucleus, the toxic action of pair cell depends on
99Tc
mIn intracellular distribution, promptly
99Tc
mBe distributed in endonuclear toxic action much larger than in cell membrane or the distribution in cytoplasm.This experiment is free in the tail vein injection reduction
99Tc
mBack 2-20 hour, the reduction of 40%-65% was free
99Tc
mBe distributed in the neoplastic cell nuclei, and vitals after one's own heart in vivo, only the reduction of 10%-18% is free for liver and kidney increased radioactivity
99Tc
mBe distributed in the nucleus.External, the reduction of weary oxygen and non-weary oxygen H460 adenocarcinoma of lung tumor cell is free
99Tc
mAt nucleus distribution no significant difference, be respectively 22% and 16%.
Coronary heart disease is the commonly encountered diseases of harm humans health, and it is that academia is thirsted for and open question always to coronary heart disease diagnosis that clinical direct non-invasive is measured ischemic myocardium.The following experimental result of the present invention shows that reduction is free
99Tc
mIt is a kind of prospect ischemic myocardium developer that has very much.
1, adopt the SD rat heart model that exsomatizes, infusion liquid adopts Ringer's solution to add glucose mM, Sodium Pyruvate mM, 12 units/L insulin, 95%O
2+ 5%CO
2Saturated.From aorta retroperfusion 15 minutes, behind the ligation left circumflex artery, the lmCi reduction was free with containing
99Tc
mWith the infusion liquid continous pouring of 0.1mCi thallium-201 30 minutes, it is free that reuse does not contain reduction
99Tc
mAnd thallium-201 (
201T1) infusion liquid perfusion 15 minutes.Heart in-20 ℃ freezing after, the minor axis cardiac muscle section of cutting about 1mm thickness along minor axis.The high-resolution parallel aperture collimator,
99Tc
mWith
201Tl double-core sketch map picture is gathered, and grand total is respectively 350K and 130K, matrix 512 * 512, Zoom3.2.The result shows that at left ventricle lower wall ischemia place, as seen very significantly reduction is free
99Tc
mRadiate dense poly-kitchen range, and
201Same area shows no obvious abnormalities on the T1 image.
2, adopt new zealand rabbit at the systemic heart ischemia model.Ketamine+penthiobarbital anesthesia, tracheal intubation is opened breast, behind the injection 1mg/kg lignocaine, the ligation left circumflex artery.Intravenous injection in 5 minutes reduction is free after the ligation
99Tc
m, after intravenous injection in 15 minutes
201Tl took out heart after 5 minutes, and with the about 50ml of cold PBS, aorta drives in the wrong direction and washes heart.Therefore, the ligation left circumflex artery caused myocardium of short duration ischemia 20 minutes.Heart in-20 ℃ freezing after, the minor axis cardiac muscle section of cutting about 1mm thickness along minor axis.The high-resolution parallel aperture collimator,
99Tc
mWith
201Tl double-core sketch map picture is gathered, and grand total is respectively 350K and 130K, matrix 512 * 512, Zoom3.2.This experiment purpose is that the demonstration reduction is free
99Tc
mCan injection back early stage (only 20 minutes) the of short duration ischemia of reflecting myocardium.Result's demonstration,
201Tl shows left circumflex artery areal area radiation defect on a large scale, point out this zone to have serious perfusion and lower, but reduction is free
99Tc
mIn this zone very strong increased radioactivity is arranged.Prompting: even very of short duration ischemia, reduction is free
99Tc
mCan show myocardial ischemia delicately in early days in injection.
Claims (8)
1, a kind of weary oxygen and neoplasm tracing agent is characterized in that with organic solvent as the dismutation reaction media, adopt Reducing agent with height
99Tc
mIt is free that hydrochlorate is reduced into stable low price
99Tc
m
2, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described organic solvent is acetonitrile or acetone.
3, by described weary oxygen of claim 2 and neoplasm tracing agent, it is characterized in that described organic solvent is an acetonitrile.
4, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described Reducing agent is stannous chloride, sodium thiosulfate, ferrous sulfate or sodium borohydride.
5, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described height
99Tc
mHydrochlorate is high
99Tc
mAcid sodium.
6, by the preparation method of described weary oxygen of claim 1 and neoplasm tracing agent, comprise the steps:
High
99Tc
mHydrochlorate is reduced into stable earlier under the Reducing agent effect
99Tc
mColloid, the latter passes through the organic solvent effect again, and it is free to form stable reduction through dismutation reaction
99Tc
m
7, the reduction of claim 1 is free
99Tc
mPurposes in the agent of preparation tumour radiotherapy localization diagnosis.
8, the reduction of claim 1 is free
99Tc
mPurposes in the radioactivity localization diagnosis agent of preparation ischemia or anoxia disease.
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现代生物技术核分子影象探针的设计与研发 曾俊,中国药物与临床,第3卷第3期 2003 * |
现代生物技术核分子影象探针的设计与研发 曾俊,中国药物与临床,第3卷第3期 2003;诊断核医学 许乐村,化学教育,第1994 4期 1994 * |
诊断核医学 许乐村,化学教育,第1994 4期 1994 * |
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