CN1544093A - Tracer agent for oxygen lacking tumor cell and its usage - Google Patents

Tracer agent for oxygen lacking tumor cell and its usage Download PDF

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CN1544093A
CN1544093A CNA2003101089048A CN200310108904A CN1544093A CN 1544093 A CN1544093 A CN 1544093A CN A2003101089048 A CNA2003101089048 A CN A2003101089048A CN 200310108904 A CN200310108904 A CN 200310108904A CN 1544093 A CN1544093 A CN 1544093A
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free
reduction
agent
cell
weary oxygen
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CN1318095C (en
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骏 曾
曾骏
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Shanghai Chest Hospital
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Shanghai Chest Hospital
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Abstract

The invention relates to a radionuclide anoxia and tumor visualization tracer agent, in particular to radionuclide visualization tracer agent deacidized free 99Tcm and deacidized free 188Re for tumor and ischemia, hypoxia diseases, wherein organic solution is used as the agent for disproportionating reaction, and reducing agents are employed to deacidize salts of high 99Tcm acid and salts of high 188Re acid into stabilized low valence free 99Tcm and free 188Re.

Description

A kind of weary oxygen and neoplasm tracing agent and uses thereof
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 0.4ml-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 0.4ml-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 99T 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 99T 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
Inject back 4 hours of injection in back 1 hour injection in back 2 hours
The free technetium of the free technetium reduction of the free technetium %ID/g of the free technetium reduction of the free technetium %ID/g of the free technetium reduction of %ID/g
Blood 4.826 5.481 blood 3.121 3.369 blood 2.435 2.449
Heart 1.562 1.957 hearts 1.468 1.082 hearts 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
Adrenal gland 10.367 12.011 adrenal gland 11.575 9.419 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 duodenums 5.319 2.589 duodenums 2.770 1.992
Small intestinal 3.137 3.562 small intestinals 3.624 3.304 small intestinals 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 ischemias
Anoxia condition A B C D E F G
Free 99Tc mO 4 -0.104 0.071 0.085 0.075 0.152 0.175 0.152
Reduction is free 99Tc m1.239 13.561 1.308 4.779 1.866 0.955 2.018
Table 3
Tumor tumor/skeletal muscle tumor/myocardium tumor/lung tumor/liver tumor/splenic tumor/kidney
18F-FMISO 0.41 2.20 2.24 1.65 0.22 ---- 0.51
123I-IAGA 2.80 3.70 ---- ---- ---- ---- ----
99Tc m-BMS 0.31 3.54 1.77 1.83 0.19 ---- 0.56
99Tc m-HL91 2.63 3.36 3.18 1.45 0.51 1.24 0.68
1 reduction is free 99Tc m3.51 38.09 5.53 7.29 1.71 5.01 2.39
2 reduction are free 99Tc m2.67 9.94 2.38 2.92 1.11 2.01 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
The back was injected 18-20 hour in 2-4 hour in the injection back
Heart 18.56% 18.94%
Liver 9.97% 12.98%
Kidney 12.05% 13.87%
HCCLM3-P15 hepatocarcinoma 43.89% 48.61%
H460 adenocarcinoma of lung 40.44% 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 m
Influence.
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 m
Influence.
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 video picture
The 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, 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 -Comparison
Human adenocarcinoma of lung H460 cell strain (providing) 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 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 1.31% ± 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.
Embodiment 2 pH values, calcium, potassium and glucose are free to anoxic cell picked-up reduction 99Tc mInfluence
Human adenocarcinoma of lung H460 cell strain (providing by U.S. The University of Texas MD AndersonCancer Center) is provided, be incubated in 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.
Embodiment 4
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.
Embodiment 6 reduction are free 99Tc mIn the body of lotus tumor nude mice, distribute
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%.
Embodiment 8, and reduction is free 99Tc mDistribution at ischemic myocardium
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 1mCi 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 ( 201Tl) 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 Tl 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 (14)

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 mHydrochlorate and height 188It is free that the Re hydrochlorate is reduced into stable low price 99Tc mWith free 188Re makes.
2, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described organic solvent be dewater and with the immiscible organic solvent of water.
3, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described organic solvent is acetonitrile or acetone.
4, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described organic solvent is an acetonitrile.
5, 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.
6, 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.
7, by described weary oxygen of claim 1 and neoplasm tracing agent, it is characterized in that described height 188The Re hydrochlorate is high 188Re acid sodium.
8, the preparation method of described weary oxygen of claim 1 and neoplasm tracing agent comprises the steps height 99Tc mHydrochlorate and height 188The Re hydrochlorate under the stannous chloride effect, is reduced into stable at Reducing agent earlier 99Tc mColloid and 188The Re colloid, the latter passes through organic solvent again, and as the acetonitrile effect, it is free to form stable reduction through dismutation reaction 99Tc mFree with reduction 188Re.
9, the method for claim 8 is during wherein the ply of paper of reduction reaction 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.
10, the method for claim 8, two kinds of reduction were free during wherein the ply of paper of dismutation reaction 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.
11, the purposes of the free technetium-99m of reduction in the agent of preparation tumour radiotherapy localization diagnosis.
12, the purposes of the free technetium-99m of reduction in preparation tumour radiotherapy video picture therapeutic agent.
13, the purposes of the free rhenium-188 of reduction in preparation tumour radiotherapy video picture therapeutic agent.
14, the purposes of the free technetium-99m of reduction in the radioactivity localization diagnosis agent of preparation ischemia, anoxia disease.
CNB2003101089048A 2003-11-27 2003-11-27 Tracer agent for oxygen lacking tumor cell and its usage Expired - Fee Related CN1318095C (en)

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