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

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

Tracer agent for hypoxic and tumor and application thereof

Technical Field

The invention belongs to the field of nuclear medicine, and particularly relates to a radionuclide hypoxic and tumor imaging tracer and application thereof in nuclide radioimaging diagnosis and treatment of tumors and hypoxic diseases.

Background

In clinical diagnosis of tumors, the nuclide radiographic imaging diagnosis has good due value. Among them, the most ideal and most widely used in clinic is fluorine-18 fluorodeoxyglucose positron emission tomography (¹⁸F-FDG PET), however,¹⁸F-FDG PET still has the following disadvantages: 1,¹⁸F-FDG PET examination is expensive, and more than 1 ten thousand RMB is required for one-time examination; 2, the positive imaging effect of pathological changes of hypermetabolic normal tissues and organs (such as brain, liver and the like) is poor; 3, high-energy positive electron nuclide¹⁸F has no therapeutic value for tumors. The examination cost of Single Photon Emission Computed Tomography (SPECT) is much lower compared to PET. Although some monodaughter nuclides also have tumor-philic characteristics, no alternative to positrons has been available to date¹⁸Single photon nuclide compounds of F-FDG. Single photon nuclide technetium-99 m (⁹⁹Tc^m) The method has the advantages of low price and convenient source, can mark a plurality of compounds, and is the nuclide which is most widely applied globally at present.⁹⁹Tc^mSome progress has been made in labeling tumor-philic compounds such as monoclonal antibodies, polypeptides, anti-tumor compounds, energy metabolism compounds, hypoxic compounds, etc., but it is still difficult to replace positrons¹⁸F-FDG. Wherein,⁹⁹Tc^mthe marked hypoxic compound has a very good application prospect, which is not only because most solid tumors have a hypoxic state, but also because tumor cells in the hypoxic state are not sensitive to radiotherapy and chemotherapy, which is a very troublesome problem in tumor treatment. Therefore, the temperature of the molten metal is controlled,⁹⁹Tc^mthe marked oxygen-deficient compound can be used for diagnosing tumor, and its Auger electron radiation characteristic or its analogue beta particle emitting nuclide rhenium-188 (R) ((R))¹⁸⁸Re) can be used for tumor therapy.

Presently preferred hypoxic imaging agents include¹⁸F-FMISO、¹²³I-IAGA、⁹⁹Tc^mNitroimi-dazole (BMS) and⁹⁹Tc^m-HL91, in the form of non-nitroimidazoles⁹⁹Tc^m-HL91 is most desirable, even if so,⁹⁹Tc^mHL91 is still difficult to be widely used in clinic.

Disclosure of Invention

The invention aims to provide a medicine for treating tumor and ischemic and anoxic diseasesHypoxic and tumor tracer for radioactive imaging diagnosis, in particular to nuclide reduction dissociation⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Re is used as hypoxic and tumor tracer. It is a further object of the invention to provide a reduced free⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Application of Re in treating tumor.

The present invention uses organic solution, such as acetonitrile, as disproportionation reaction medium, and adopts reducing agent, such as stannous chloride⁹⁹Tc^mAcid salt (sodium) and sodium¹⁸⁸Re acid salt (sodium) reduction to stable low valence free⁹⁹Tc^mAnd free¹⁸⁸Re and experimentally confirmed that free radicals are reduced⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Re is a very ideal hypoxic and tumor tracer, and can be used for radioactive imaging diagnosis and treatment of tumors and diagnosis of other ischemic and hypoxic diseases (such as ischemic heart disease).

The organic solvent is water and water immiscible organic solvent such as ethyl acetate, chloroform and the like, such as acetonitrile, acetone and the like, and the reducing agent is stannous chloride, sodium thiosulfate, ferrous sulfate, sodium borohydride and the like.

The invention is realized by the following method

First, preparation of reduced free⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Re

Using organic solvent as medium, and reducing agent (such as stannous chloride) to increase the content of the organic solvent⁹⁹Tc^mAcid salt (sodium) and sodium¹⁸⁸Re acid salt (sodium) reduction to stable low valence free⁹⁹Tc^mAnd free¹⁸⁸Re。

Height of⁹⁹Tc^mAcid salt of (A)⁹⁹Tc^mO₄ ^-) And height¹⁸⁸Re acid salt of (A)¹⁸⁸ReO₄ ^-) Reducing into stable form under the action of reducing agent, such as stannous chloride, sodium thiosulfate, ferrous sulfate, etc⁹⁹Tc^mA colloid and¹⁸⁸re colloid, the latter being further organosolvedThe agent, such as acetonitrile, acetone, etc., forms two stable reduced free radicals through disproportionation reaction⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Re。

Two, high⁹⁹Tc^mAcid (A), (B)⁹⁹Tc^mO₄ ^-) By reduction reaction of⁹⁹Tc^mDisproportionation of colloid

1, reduction reaction: in a sterile vacuum flask, 0.1ml of⁹⁹Tc^mO₄ ^-(about 148MBq), 15. mu.l of a reducing solution containing 150ug of stannous chloride was added thereto, and the reaction was carried out for 5min at room temperature. The analysis is carried out by adopting paper chromatography and silica gel plate chromatography, and the developing agents are water and acetone respectively. In paper chromatography⁹⁹Tc^mO₄ ^-And⁹⁹Tc^mthe Rf values of the colloid are 0.70-0.75 and 0.00-0.10 respectively; chromatographed on silica gel plate⁹⁹Tc^mO₄ ^-And⁹⁹Tc^mthe Rf values of the colloids are 0.97-1.00 and 0.00-0.10, respectively. The results showed that 100% of⁹⁹Tc^mO₄ ^-Is reduced into⁹⁹Tc^mAnd (3) colloid.

2, disproportionation reaction: in a sterile vacuum flask, 0.1ml of⁹⁹Tc^mAfter the reaction, 0.4ml to 0.6ml of water or 0.4ml to 0.6ml of organic solvent is added for reaction for 25min at room temperature. Paper chromatography and silica gel plate chromatography are adopted, and developing agents are water and acetone respectively. Two kinds of reduction free in paper chromatography⁹⁹Tc^mHas Rf values of 0.80-0.85 and 0.70-0.75 respectively,⁹⁹Tc^mthe Rf value of the colloid is 0.00-0.10; two kinds of reduction free in silica gel plate chromatography⁹⁹Tc^mHas Rf values of 0.95-1.00 and 0.80-0.85,⁹⁹Tc^mthe colloid has an Rf value of 0.00 to 0.10. The results show that the disproportionation capability of the organic solvent on the colloid technetium is respectively 96.61 percent of acetone, 94.00 percent of acetonitrile, 90.20 percent of tetrahydrofuran, 86.49 percent of dichloromethane, 84.83 percent of acetic acid, 68.58 percent of ethanol, 67.95 percent of N-hexane, 59.68 percent of toluene, 57.83 percent of N, N-dimethylformamide, 47.22 percent of ethyl acetate, 44.15 percent of trichloromethane and 42.71 percent of methanolDiethyl ether 37.78% and water 0.00%. In the tested organic solvent, acetone, acetonitrile and tetrahydrofuran make more than 90 percent⁹⁹Tc^mDisproportionation of colloid into reduction dissociation⁹⁹Tc^mAnd the water is to⁹⁹Tc^mThe colloid has no disproportionation effect.

Thirdly, selective preparation of reduced free⁹⁹Tc^mOrganic solvent (2)

From the above disproportionation reaction test, it was found that the pair of acetone, acetonitrile and tetrahydrofuran⁹⁹Tc^mThe disproportionation of the colloid is strongest, which still does not determine that acetone, acetonitrile and tetrahydrofuran are the best preparation stability reducing free⁹⁹Tc^mSince the reducing agent is always present in the actual operation, the influence of the reducing agent on the disproportionation reaction still needs to be considered. Therefore, the invention continues the reduction-disproportionation-reducibility experiment, the reduction-disproportionation-reducibility experiment: in a sterile vacuum, 0.4ml to 0.6ml of water or the following organic solvent is added, 15 mu l of freshly prepared reducing solution containing 150ug of stannous chloride is added, and 0.1ml of reducing solution containing 150ug of stannous chloride is added⁹⁹Tc^mO₄ ^-(about 148MBq), reacted at room temperature for 15min, and finally added 0.2ml of water containing excess stannous chloride (5 mg/ml). The results showed that the organic solvents having the disproportionation ability from strong to weak were acetonitrile 91.04%, N-dimethylformamide 54.89%, acetone 51.01%, acetic acid 38.03%, methanol 34.47%, tetrahydrofuran 33.61%, dichloromethane 31.77%, toluene 25.87%, diethyl ether 23.38%, N-hexane 15.33%, chloroform 13.91%, ethanol 12.19%, ethyl acetate 7.06% and water 0.00%, respectively.

In the tested organic solvent, the acetonitrile accounts for more than 90 percent⁹⁹Tc^mDisproportionation of colloid into reduction dissociation⁹⁹Tc^mThe organic solvent is preferably reduced. The disproportionation of DMF and acetone is about 50%, the disproportionation of other organic solvents is below 40%, and water acts on⁹⁹Tc^mThe colloid has no disproportionation effect.

Fourthly, reduction dissociation⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Re

The method comprises the following steps: in a sterile vacuum flask, according to⁹⁹Tc^mO₄ ^-Or¹⁸⁸ReO₄ ^-Adding a proper amount of organic solvent with strong disproportionation, such as acetonitrile, into the eluent according to the actual input amount: the volume of the leacheate is more than 3: 1. Adding 50-250 ug of freshly prepared reducing agent, such as stannous chloride, and immediately adding fresh⁹⁹Tc^mO₄ ^-Or¹⁸⁸ReO₄ ^-Leacheate, room temperature reaction for at least 15 minutes. And (3) completely evaporating and pumping the organic solvent and the water in the sterile vacuum bottle by using a negative subpump until a trace of off-white solid appears at the bottom of the bottle. The white solid was dissolved thoroughly with sterile distilled water and used. The radioactive yield obtained is greater than 85%.

The method 2 comprises the following steps: adding 50-250 ug of fresh reducing agent such as stannous chloride, and fresh into sterile vacuum bottle⁹⁹Tc^mO₄ ^-Or¹⁸⁸ReO₄ ^-Eluting, and adding appropriate amount of organic solvent such as acetonitrile, wherein the volume of the organic solvent to the volume of the eluting solution is more than 3: 1. The reaction was carried out at room temperature for at least 15 minutes. And (3) completely evaporating and pumping the organic solvent and the water in the sterile vacuum bottle by using a negative subpump until a trace of off-white solid appears at the bottom of the bottle. The white solid was dissolved thoroughly with sterile distilled water and used as soon as possible. The radioactive yield obtained is greater than 85%.

Fifthly, dissociating⁹⁹Tc^mO₄ ^-And reduction of free radicals⁹⁹Tc^mComparison of physical, chemical and biological properties 1, comparison of physical properties: thin Layer Chromatography (TLC), paper electrophoresis and reverse gradient High Performance Liquid Chromatography (HPLC) can be used to separate two kinds of reduction dissociation⁹⁹Tc^mOne of them is 80% -90% and free⁹⁹Tc^mO₄ ^-Cannot be distinguished from each other. Reducing free radical⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Electrophoresis from cathode to anode with voltage of 300V and electrophoresis timeFor 30 minutes, the electrolyte was PBS (PH 7.4). Reducing free radical⁹⁹Tc^m(left side) and free⁹⁹Tc^mO₄ ^-(Right side) all crawled the anode, indicating reductive dissociation⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Are both anionic. Wherein the free radical is reduced⁹⁹Tc^mTwo kinds of the materials are available, one of the materials accounts for 80% -90%, and the materials are free⁹⁹Tc^mO₄ ^-Cannot be distinguished from each other, has a creeping distance of about 3cm, and the other is free⁹⁹Tc^mO₄ ^-Different, the creep distance is about 6cm and accounts for 10-20%. 2, comparison of chemical characteristics: will dissociate⁹⁹Tc^mO₄ ^-And reduction of free radicals⁹⁹Tc^mRespectively adding the mixture into a solution containing a sulfhydryl compound, such as dimercaptoethanol, and carrying out labeling reaction for 15 minutes at room temperature under the labeling reaction condition 1 of PBS; labeling reaction condition 2 was 25mM lactic acid; labeling reaction condition 3 was 65mM lactic acid; the labeling reaction condition 4 was 1mM CaCl₂+6mM NaCl; the labeling reaction condition 5 was 6mM CaCl₂+6mM NaCl; the labeling reaction condition 6 was 65mM lactic acid +6mM CaCl₂+6mM NaCl. The results show that lactic acid does not promote reductive dissociation under aerobic and anaerobic conditions⁹⁹Tc^mCombined with dimercaptopropane sodium sulfonate, calcium ion can promote reduction dissociation under anaerobic condition⁹⁹Tc^mThe combination with dimercaptopropane sodium sulfonate can increase the combination rate by more than two times, and the combination rate is obviously higher than that in an aerobic state. The results show that⁹⁹Tc^mO₄ ^-Can not mark any sulfhydryl-containing compound and reduce the free⁹⁹Tc^mCan mark the sulfhydryl-containing compound with the marking rate of 10-70 percent. Illustrating the reduction of free radicals⁹⁹Tc^mIs active in chemical nature. Proved that the clinical sulfydryl-containing detoxification drug can inhibit the in vivo tumor uptake reduction dissociation⁹⁹Tc^m。

Comparison of biological Properties: (1) respectively injecting the free compound from tail vein of nude mouse⁹⁹Tc^mO₄ ^-And reduction of free radicals⁹⁹Tc^m1 part after injection,At 2 and 4 hours, the in vivo profiles of the two compounds were different, with significant differences in liver, lung, spleen, pancreas, kidney, stomach, colon and thyroid profiles. (2) Reducing free radical⁹⁹Tc^mHas affinity effect on hypoxic cells or tissues and can be dissociated⁹⁹Tc^mO₄ ^-Has no affinity effect on hypoxic cells or tissues. Table 1 shows the in vivo distribution of the two compounds, Table 2 shows the reduced free form⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Has affinity effect on hypoxic cells or tissues.

Sixthly, reduction dissociation⁹⁹Tc^mAffinity and dissociation for hypoxic cells or tissues⁹⁹Tc^mO₄ ^-Comparison of

Ischemia and hypoxia of cells or tissues can cause a series of metabolic abnormalities due to reduction and dissociation⁹⁹Tc^mThe following metabolic abnormalities caused by ischemia hypoxia may change the reduction liberation of ischemia hypoxia cells (or tissues)⁹⁹Tc^mThe intake of (1). The abnormal metabolism includes cellular hypoxia of local tissues, increase of osmotic pressure, decrease of pH value, accumulation of extracellular potassium ions, overload of intracellular calcium ions, generation of oxygen free radicals, and the like. The results of the invention prove that the H460 lung cancer cells can be promoted to take up, reduce and dissociate by the hypoxic oxygen, the PH value reduction, the extracellular potassium ion accumulation and the intracellular calcium ion overload⁹⁹Tc^mWhile hypoxia and PH value decrease inhibit H460 lung cancer cell from absorbing free cells⁹⁹Tc^mO₄ ^-. H460 lung cancer cell pair reduction dissociation under normal, extracellular potassium ion accumulation, hypoxia and PH value reduction states⁹⁹Tc^mRespectively, the intake amounts of⁹⁹Tc^mO₄ ^-11.9, 13.3, 191.0 and 63.7 times of the cell intake, wherein the hypoxia is the cell intake of reduction free cells for determining ischemia hypoxia⁹⁹Tc^mThe most important factor of (c). In addition, the reduction of pH and the overload of calcium ions in the hypoxia state of the cells influence the uptake and reduction of free cells⁹⁹Tc^mAre important factors of.

The experiment of the invention proves that the free is reduced⁹⁹Tc^mThe distribution indexes in vivo are obviously superior to other hypoxia imaging agents, and the tumor imaging effect is ideal. Reducing free radical⁹⁹Tc^mIn the distribution of tumor subcellular cells, 40-65% of the tumor subcellular cells are distributed in cell nucleus, and the reduction dissociation can be obviously increased⁹⁹Tc^mToxic and killing effects on hypoxic tumor cells, and in addition, free¹⁸⁸Re is a therapeutic nuclide that emits beta particles with a physical half-life of up to 17 hours, whereas radioactivity has been concentrated primarily in hypoxic tumors 17 hours after intravenous injection, thus reducing free radicals¹⁸⁸Re has high curative effect on tumor. Reducing free radical⁹⁹Tc^mCan also be used for diagnosing and evaluating ischemic diseases, such as coronary heart disease.

Table 1 is free⁹⁹T^mO₄ ^-And reduction of free radicals⁹⁹Tc^mIn vivo distribution at 1, 2 and 4 hours post injection.

Table 2 shows the reduction dissociation⁹⁹Tc^mAnd free⁹⁹T^mO₄ ^-Comparison of% cell binding at different metabolic states.

Table 3 shows the reduction dissociation⁹⁹Tc^mCompared to the in vivo distribution of known hypoxic imaging agents.

Table 4 shows the reduction dissociation⁹⁹Tc^mAfter in vivo injection, the distribution of nuclei in normal tissues and nuclei in tumor-bearing cells in nude mice were compared.

TABLE 1

1 hour after injection and 2 hours after injection and 4 hours after injection

% ID/g free technetium reduced free technetium

Blood 4.8265.481 blood 3.1213.369 blood 2.4352.449

Heart 1.5621.957 heart 1.4681.082 heart 0.8090.966

Liver disease 3.678 9.323 Liver disease 2.413 6.758 Liver disease 2.500 6.641

Lung (lung) 3.162 11.953 Lung (lung) 1.954 4.971 Lung (lung) 1.230 4.22

Thymus 1.6363.133 thymus 2.5051.291 thymus 1.0731.245

Spleen 2.420 4.455 Spleen 0.795 3.849 Spleen 1.006 4.016

Pancreas gland 5.011 1.764 Pancreas gland 2.164 1.205 Pancreas gland 1.903 0.756

Kidney (Kidney) 2.883 7.720 Kidney (Kidney) 1.976 7.171 Kidney (Kidney) 1.700 7.409

Adrenal gland 10.36712.011 adrenal gland 11.5759.419 adrenal gland 3.3158.492

Antrum of stomach 14.65 16.368 Antrum of stomach 24.48 14.63 Antrum of stomach 12.24 7.155

Stomach body 6.820 9.689 Stomach body 14.52 6.761 Stomach body 17.13 6.862

Duodenum 3.2765.183 duodenum 5.3192.589 duodenum 2.7701.992

Small intestine 3.1373.562 small intestine 3.6243.304 small intestine 2.3172.270

Colon 2.064 1.179 Colon 7.291 1.007 Colon 1.717 0.299

Skeletal muscle 1.7281.591 skeletal muscle 0.5980.68501 skeletal muscle 0.5950.651

Thyroid gland 72.01 92.518 Thyroid gland 123.5 47.71 Thyroid gland 82.27 21.63

TABLE 2

Different ischemia

Hypoxic conditions AB C D E F G

Free form⁹⁹Tc^mO₄ ^- 0.104 0.071 0.085 0.075 0.152 0.175 0.152

Reducing free radical⁹⁹Tc^m 1.239 13.561 1.308 4.779 1.866 0.955 2.018

TABLE 3

Tumor/hematological tumor/skeletal muscle tumor/myocardial tumor/lung tumor/liver tumor/spleen tumor/kidney tumor

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 of free radical⁹⁹Tc^m 3.51 38.09 5.53 7.29 1.71 5.01 2.39

2 reduction swimmingSeparation device⁹⁹Tc^m 2.67 9.94 2.38 2.92 1.11 2.01 0.92

Wherein 1 is reduced to free⁹⁹Tc^mIs human liver cancer HCCLM3-P15,

2 reduction of free radical⁹⁹Tc^mIs human H460 lung adenocarcinoma.

TABLE 4

2-4 hours after injection and 18-20 hours after injection

Heart 18.56% 18.94%

9.97% of liver and 12.98%

Kidney 12.05% 13.87%

HCCLM3-P15 liver cancer 43.89% 48.61%

H460 Lung adenocarcinoma 40.44% 63.10%

Drawings

FIG. 1, preparation of reduced free⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Technical roadmap for Re

FIG. 2 uptake of reduced dissociative H460 lung adenocarcinoma cells by hypoxic and simulated ischemic metabolic changes⁹⁹Tc^mIs/are as follows

Influence.

FIG. 3 lactic acid vs. oxygen andhypoxic H460 lung adenocarcinoma cell uptake reduction dissociation⁹⁹Tc^mAnd free

⁹⁹Tc^mO₄ ^-Comparison of the effects.

FIG. 4 reduction of free uptake by hypoxic H460 lung adenocarcinoma cells by lactate, potassium, calcium and glucose concentrations⁹⁹Tc^m

The influence of (c).

FIG. 5, an example of HCP-⁹⁹Tc^mAnd (5) displaying the result.

FIG. 6 shows an example of the free form of HCC-M3-P15 HCC in nude mouse by intravenous injection⁹⁹Tc^mSubsequent development

And (6) obtaining the result.

FIG. 7 shows the perfusion results of isolated SD rat heart model, in which the left arrow indicates the ischemia of the lower side wall of the left ventricle of the SD rat heart, and the obvious reduction dissociation is observed⁹⁹Tc^mRadiation concentration range, right side²⁰¹No obvious abnormality is found in the same part of the Tl image.

FIG. 8 is a graph showing transient myocardial ischemia in the left circumflex branch region of coronary artery of New Zealand rabbit, in which the arrow on the right side indicates²⁰¹Tl images show extensive radioactive defects in the area, reducing the free⁹⁹Tc^mIn the early stage of injection, myocardial ischemia is sensitively shown.

FIG. 9, reduction dissociation⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The paper electrophoresis analysis of (1) was compared.

FIG. 10, reduction dissociation⁹⁹Tc^mA comparison of sodium dimercaptopropanesulfonate labeled in both aerobic and anaerobic states.

Detailed Description

Example 1 in vitro uptake of reducing free by aerobic and hypoxic cells⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Comparison of

Human lung adenocarcinoma H460 cell line (supplied by The University of Texas MD Anderson cancer Center, USA) was cultured in 24-well culture plate with 1640 culture medium, H460 cells were in growth and proliferation stage, and free cells were reduced under The following different experimental conditions⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-In vitro cell binding experiments. The results show that H460 lung cancer cells reduce free cells under the conditions of normal, extracellular potassium ion accumulation, hypoxia and PH value reduction⁹⁹Tc^mRespectively, the intake amounts of⁹⁹Tc^mO₄ ^-11.9, 13.3, 191.0 and 63.7 times of the cell intake, wherein the hypoxia is the cell intake of reduction free cells for determining ischemia hypoxia⁹⁹Tc^mThe most important factor of (c). In addition, the reduction of pH value and the overload of calcium ions of the cells in the hypoxia state influence the uptake of reduction dissociation by the hypoxia cells⁹⁹Tc^mAre important factors of. The experimental conditions were as follows:

condition a: replacing fresh 1640 culture solution 0.5ml, adding reduction dissociation⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) is 1.24% +/-0.26% and 0.11% +/-0.02%, respectively, reducing free⁹⁹Tc^mThe H460 lung cancer cell intake is⁹⁹Tc^mO₄ ^-11.9 times the amount of cells taken up.

Condition B: replacing fresh 1640 culture solution 0.5ml, adding reduction dissociation⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, after negative pressure was applied for 15 minutes, H460 cells were hypoxic and cell-binding reaction was performed at 37 ℃ for 3 hours. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) is 13.56% + -0.15% and 0.07% + -0.002%, respectively, reducing the free⁹⁹Tc^mThe H460 lung cancer cell intake is⁹⁹Tc^mO₄ ^-191.0 times of the cell uptake.

Condition C: changing fresh 1640 culture solution containing 350mM sucrose (osmotic pressure of 380mOsm), and adding reduced free⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) is 1.31% + -0.19% and 0.07% + -0.006%, respectively, reducing free⁹⁹Tc^mThe H460 lung cancer cell intake is⁹⁹Tc^mO₄ ^-18.7 times the amount of cells taken up.

Condition D: replacing with 55mM succinic acid or lactic acid (pH 4.5) fresh 1640 culture medium, and adding reducing free medium⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) was 4.78% + -0.47% and 0.07% + -0.009%, respectively, and free reduction⁹⁹Tc^mThe H460 lung cancer cell intake is⁹⁹Tc^mO₄ ^-The cell uptake was 63.7 times that of the cells.

Condition E: replacing the fresh 1640 culture solution containing 0.5mM CaCl2+9mM NaCl, and adding the reduction free⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) is 1.87% +/-0.48% and 0.10% +/-0.05%, respectively, reducing free⁹⁹Tc^mH460 Lung cancer cell uptake ofGet the amount of⁹⁹Tc^mO₄ ^-18.7 times the amount of cells taken up.

Condition F: changing fresh serum-removed 1640 culture solution containing 0.15% hydrogen peroxide and 20 micrograms of copper sulfate, adding reduced free⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) is 0.96% + -0.17% and 0.18% + -0.06%, respectively, reducing free⁹⁹Tc^mThe H460 lung cancer cell intake is⁹⁹Tc^mO₄ ^-5.4 times of the cell uptake.

Condition G: replacing the fresh 1640 culture solution containing 10mM KCl and adding the reduction free⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Then, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, the free radicals are reduced⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-The% cellular uptake of (D) was 2.02% +/-0.08% and 0.15% +/-0.01%, respectively, and free reduction⁹⁹Tc^mThe H460 lung cancer cell intake is⁹⁹Tc^mO₄ ^-13.3 times the amount of cells taken up.

Example 2 uptake of reduced free form by pH, calcium, potassium and glucose into hypoxic cells⁹⁹Tc^mInfluence of (2)

Human lung adenocarcinoma H460 cell line (supplied by The University of Texas MD Anderson cancer Center, USA) was cultured in a 24-well culture plate with 1640 culture solution, H460 cells were in growth and proliferation stage, and negative pressure was applied for 20-30 min to cause hypoxia. The results show that in the presence of oxygen, the cells take up and release as the pH decreases⁹⁹Tc^mO₄ ^-Gradually decreases, and on the contrary, when the pH value drops to 5.5, the aerobic cells take up and reduce the free⁹⁹Tc^mInitially increasing as the PH decreases. In thatIn the case of hypoxia, the cells take up and reduce the dissociation⁹⁹Tc^mIs more sensitive to pH change, and when the pH value is reduced to 7.0, the anaerobic cells take up and reduce the free oxygen⁹⁹Tc^mThat is, the pH value is increased along with the decrease of the pH value, which shows that the pH value change of the local microenvironment can obviously influence the anaerobic cells to reduce and dissociate⁹⁹Tc^mThe anaerobic glycolytic metabolism of the anaerobic cells is enhanced and the pH value of the local microenvironment is reduced so as to facilitate the anaerobic cell to take up and reduce the free oxygen⁹⁹Tc^m。

The hypoxia of cells is accompanied by the overload of calcium ions in cells and the accumulation of potassium outside the cells, and the experiment proves that in the presence of calcium, hypoxic cells take up and reduce free⁹⁹Tc^mThe obvious increase indicates that calcium overload causes the uptake and reduction liberation of anaerobic cells⁹⁹Tc^mIs another important factor. Increased concentrations of potassium and glucose promote uptake of reduced free hypoxic cells⁹⁹Tc^mHas no obvious effect and specificity.

Example 3 uptake of reduced free by hypoxic cells⁹⁹Tc^mMechanism of (1) experiment

Due to reduction of free radicals⁹⁹Tc^mThe anion transport (anion transporter) of the cell membrane may be involved in reducing the free radical⁹⁹Tc^mBecause the anion transporter is normally inactive, but when the pH is lowered, the cell membrane anion transporter can be activated, thereby facilitating the reduction of free ions⁹⁹Tc^mEnter hypoxic cells.

With chemically inactive free radicals⁹⁹Tc^mO₄ ^-In contrast, reduced free⁹⁹Tc^mHas chemical activity, and can be combined with macromolecular compound containing sulfhydryl and amino, thereby remaining in cells. The following experiment proves that the hypoxia can promote the reduction dissociation⁹⁹Tc^mCombined with macromolecular compounds containing sulfhydryl and amino.

The dimercaptopropane sodium sulfonate contains twoSulfhydryl group, which can be combined with metal, is a metal poisoning antidote used in clinic. Due to reduction of free radicals⁹⁹Tc^mHas chemical activity and can be combined with dimercaptopropane sodium sulfonate. Under the aerobic and anaerobic conditions, lactic acid can not promote reductive dissociation⁹⁹Tc^mThe combination with dimercaptopropane sodium sulfonate shows that the acidic environment promotes the uptake, reduction and dissociation of cells⁹⁹Tc^mAnd reduction dissociation of⁹⁹Tc^mThe labeling of macromolecular compounds containing sulfhydryl groups and amino groups is not directly related. However, calcium ions promote reductive dissociation in the anoxic state⁹⁹Tc^mThe combination with dimercaptopropane sodium sulfonate can increase the combination rate by more than two times, and the combination rate is obviously higher than that in an aerobic state. Illustrating that calcium ions promote the uptake and reduction of hypoxia cells⁹⁹Tc^mAnd reduction dissociation of⁹⁹Tc^mThe capability of marking macromolecular compounds containing sulfydryl and amino is strengthened in a certain relation.

The results demonstrate that the acidic environment generated by hypoxic cells contributes to reducing dissociation⁹⁹Tc^mEnter the cell, and hypoxia-induced intracellular calcium overload helps to reduce dissociation⁹⁹Tc^mCombined with macromolecular compounds containing sulfhydryl and amino.

Example 4

Cellular membrane potential changes and cellular membrane integrity reduction of cell uptake⁹⁹Tc^mInfluence of (2)

Whether physiological and pathological cell membrane potential changes, or whether hypoxia or ischemia-induced cell necrosis or cell membrane destruction takes up or reduces cell dissociation⁹⁹Tc^mHas an influence on the determination of the free reduction⁹⁹Tc^mIt is important whether it is a hypoxic tracer. Human lung adenocarcinoma H460 cell line (supplied by The University of Texas MDAnderson Cancer Center, USA) is cultured in 24-well culture plate with 1640 culture solution, H460 cell is in growth and proliferation stage, and RPMI1640 culture solution is replaced by cell membrane depolarization solution (containing 10mM KCl), cell membrane hyperpolarization solution (containing 1mM sodium tetraphenylborate) and cell membrane destructive solution (containing 0.5% Triton X-100). Adding reducing free radical⁹⁹Tc^mThen, the reaction was performed in a carbon dioxide incubator for 3 hours in combination with H460 cells. As a result, normal control, cell membrane depolarization, cell membrane hyperpolarization, and reduced liberation following cell membrane disruption⁹⁹Tc^mThe cell binding rates were 0.807% + -0.062%, 1.614% + -0.249%, 2.817% + -1.608% and 0.272% + -0.019%, respectively. Wherein cell membrane disruption causes cellular uptake and reductive liberation⁹⁹Tc^mThe obvious decrease indicates that necrotic cells do not take up and reduce free cells⁹⁹Tc^m. Can make cell take up and reduce to free whether cell membrane depolarization or hyperpolarization⁹⁹Tc^mThere is an increase.

Example 5 reduction of free⁹⁹Tc^mAnd free⁹⁹Tc^mO₄ ^-Distribution comparison in normal nude mice

Nude mice (23-27 g body weight) are fasted for 6-12 hours, and are reduced and dissociated by tail vein injection under the waking state⁹⁹Tc^mOr free from⁹⁹Tc^mO₄ ^-About 15. mu. Ci (555Bq) and injection volume less than 0.2 ml. Nude mice were sacrificed at 1, 2 and 4 hours after injection, blood and each tissue organ were taken to measure the per gram tissue count and the total injection count, and the percentage of the per gram tissue count to the total injection count (% ID/g) was calculated, and the results showed that free reduction⁹⁹Tc^mThe radioactivity distribution in the liver, lung, spleen and kidney is significantly higher than that in the free form⁹⁹Tc^mO₄ ^-(ii) a In contrast, 2 hours after injection, free was reduced⁹⁹Tc^mThe radioactivity distribution in pancreas, stomach, colon and thyroid is significantly lower than that in the free form⁹⁹Tc^mO₄ ^-. The results suggest that the free form is reduced⁹⁹Tc^mIs easily taken up by normal tissues and organs and can be dissociated⁹⁹Tc^mO₄ ^-More prone to excretion from the digestive tract. The results of this example are consistent with the results of in vitro cell binding experiments, in which aerobic cells are released by reduction⁹⁹Tc^mThe intake is free⁹⁹Tc^mO₄ ^-11.9 times of.

Example 6 reduction of free⁹⁹Tc^mDistribution in vivo in tumor-bearing nude mice

Human hepatoma HCCLM3-P15 cell line (purchased from liver cancer research institute of Zhongshan Hospital, University of double denier) and human H460 lung adenocarcinoma cell line (supplied by The University of Texas MD Anderson cancer center, USA) were cultured in RPMI1640 medium (SIGMA product) containing calf serum. The proliferating tumor cells were inoculated in the right underarm of nude mice (purchased from shanghai institute for tumor). After 4-10 weeks after inoculation, the tumor grows to be more than 0.5cm for use. The tumor-bearing nude mice are reduced and dissociated by tail vein injection in a clear-headed state⁹⁹Tc^mAbout 250. mu. Ci (9250Bq) and injection volume less than 0.2 ml. Anesthetization was performed 2, 4, 8, 12 and 18 hours after injection, tumor-bearing nude mice were sacrificed after imaging, blood, tumor and various tissue organs were taken to measure the counts per gram of tissue and the total counts injected, the percentage of the counts per gram of tissue to the total counts injected (% ID/g) was calculated, and finally the% ID/g ratio of tumor and normal tissues was calculated. The results show that the free radicals are reduced⁹⁹Tc^mThe ratio of the percent ID/g of the tumor, blood, skeletal muscle, heart, lung, liver, spleen and kidney is obviously better than that of other hypoxia imaging agents, including the best hypoxia imaging agent at present⁹⁹Tc^m-HL 91. As a result, it was confirmed that the free radical was reduced⁹⁹Tc^mHas excellent characteristics of being philic to hypoxic tumors, is a breakthrough progress of hypoxic imaging research, and reduces dissociation⁹⁹Tc^mWill become the most ideal hypoxia imaging agent. Nude mouse tail vein injection reduction free of H460 lung adenocarcinoma⁹⁹Tc^mImaging was performed 2, 6 and 18 hours after injection, and tumors were clearly visualized 2 hours after injection (with necrotic foci in the center), 18 hours with the major radioactive dose concentrated in the tumor tissue and only a small amount of radioactivity remaining in the liver and stomach. HCC-M3-P15 liver cancer nude mouse tail vein injection reduction free form of lotus⁹⁹Tc^mImaging at 4, 8 and 20 hours later, tumors were approximately 0.7 × 0.7 × 0.4cm, and tumors clearly showed strong radioactivity distribution in thyroid, stomach and liver.

Example 7 reductive dissociation⁹⁹Tc^mIn tumor and normalSubcellular distribution of tissue

⁹⁹Tc^mIs an Auger electron radionuclide, and has toxic effect on cells dependent on Auger electron radionuclide⁹⁹Tc^mDistribution within the cell, i.e.⁹⁹Tc^mThe toxic effects distributed in the nucleus are much greater than in the cell membrane or in the cytoplasm. This experiment was performed by tail vein injection to reduce dissociation⁹⁹Tc^mAfter 2-20 hours, 40% -65% of reduction dissociation⁹⁹Tc^mThe radioactive substance is distributed in tumor cell nucleus, while the radioactive substance is distributed in important organs such as heart, liver and kidney by only 10% -18% of reduced free substance⁹⁹Tc^mDistributed in the nucleus. In vitro, reduced dissociation of hypoxic and non-hypoxic H460 Lung adenocarcinoma tumor cells⁹⁹Tc^mThere was no significant difference in nuclear distribution, 22% and 16%, respectively.

Example 8 reductive liberation⁹⁹Tc^mDistribution in ischemic myocardium

Coronary heart disease is a common disease which endangers human health, and the diagnosis of coronary heart disease by directly and clinically non-invasively measuring ischemic myocardium is a problem which is always desired and not solved by the academia. The following experimental results show that the free radicals are reduced⁹⁹Tc^mIs a promising myocardial ischemia imaging agent.

1, adopting an isolated SD rat heart model, adding glucose mM, sodium pyruvate mM, 12 units/L insulin and 95% O into perfusate by adopting ringer's solution₂+5％CO₂And (4) saturation. Retrograde perfusion from aorta for 15min, ligation of left circumflex coronary artery, reduction of free with 1mCi⁹⁹Tc^mAnd 0.1mCi thallium-201 for 30 minutes, and then no reducing dissociation⁹⁹Tc^mAnd thallium-201 (²⁰¹Tl) for 15 minutes. After freezing the heart at-20 ℃, short axis myocardial sections of approximately 1mm thickness were cut along the short axis. A high-resolution parallel-hole collimator, a collimator,⁹⁹Tc^mand²⁰¹tl dual-core pixel image acquisition, total count is 350K and 130K respectively, matrix 512 x 512, zoom 3.2. The results show that under the left ventricleIn the side wall ischemia, very obvious reductive dissociation can be seen⁹⁹Tc^mA radiation concentration range, and²⁰¹no obvious abnormality is seen at the same position on the Tl image.

2, New Zealand rabbit in vivo cardiac ischemia model was used. Ketamine + thiopentan sodium anesthesia, tracheal intubation, thoracotomy, injection of 1mg/Kg lidocaine, ligation of the left circumflex coronary artery. 5 minutes after ligation, the free was reduced by intravenous injection⁹⁹Tc^mAnd then the intravenous injection is carried out for 15 minutes²⁰¹Tl, and the heart was removed over 5 minutes and flushed retrograde with cold PBS approximately 50ml of aorta. Therefore, ligation of the left circumflex coronary artery resulted in transient myocardial ischemia for 20 minutes. After freezing the heart at-20 ℃, short axis myocardial sections of approximately 1mm thickness were cut along the short axis. A high-resolution parallel-hole collimator, a collimator,⁹⁹Tc^mand²⁰¹tl dual-core pixel image acquisition, total count is 350K and 130K respectively, matrix 512 x 512, zoom 3.2. The purpose of this experiment was to demonstrate reductive dissociation⁹⁹Tc^mWhether transient ischemia of the myocardium can be reflected early (only 20 minutes) after injection. The results show that it is possible to display,²⁰¹tl showed extensive radiation defect in the region of the left circumflex branch of the coronary artery, suggesting that severe perfusion reduction, but reduced free⁹⁹Tc^mThere is a very strong radioactivity distribution in this region. And (4) prompting: even in very transient ischemia, the free radicals are reduced⁹⁹Tc^mMyocardial ischemia can be sensitively shown in the early stage of injection.

Claims (14)

1. A tracer agent for hypoxic and tumor features that organic solvent is used as disproportionation medium and reducing agent is used to increase the oxygen content in tumor⁹⁹Tc^mAcid salts and higher¹⁸⁸Reduction of Re acid salt to stable low valence free⁹⁹Tc^mAnd free¹⁸⁸And Re.

2. The hypoxic and tumor tracer of claim 1, wherein said organic solvent is a water-removing and water-immiscible organic solvent.

3. The hypoxic and tumor tracer according to claim 1, characterized in that the organic solvent is acetonitrile or acetone.

4. The hypoxic and tumor tracer of claim 1, wherein said organic solvent is acetonitrile.

5. The hypoxic and tumor tracer of claim 1, wherein said reducing agent is stannous chloride, sodium thiosulfate, ferrous sulfate, or sodium borohydride.

6. The hypoxic and tumor tracer of claim 1, wherein said high level is⁹⁹Tc^mThe acid salt is high⁹⁹Tc^mSodium salt.

7. The hypoxic and tumor tracer of claim 1, wherein said high level is¹⁸⁸Re acid salt is high¹⁸⁸Re sodium salt.

8. The method of claim 1, comprising the step of⁹⁹Tc^mAcid salts and higher¹⁸⁸Re acid salt is reduced to stable under the action of reducing agent, such as stannous chloride⁹⁹Tc^mA colloid and¹⁸⁸re colloid, which is reacted with an organic solvent, such as acetonitrile, via disproportionation to form stable reduced free⁹⁹Tc^mAnd reduction of free radicals¹⁸⁸Re。

9. The method of claim 8, wherein the reduction reaction is in paper chromatography⁹⁹Tc^mO₄ ^-And⁹⁹Tc^mthe Rf values of the colloid are 0.70-0.75 and 0.00-0.10 respectively; chromatographed on silica gel plate⁹⁹Tc^mO₄ ^-And⁹⁹Tc^mthe Rf values of the colloids are 0.97-1.00 and 0.00-0.10, respectively.

10. The method of claim 8 wherein two of the reduced freeforms are present in a paper chromatography step of disproportionation⁹⁹Tc^mHas Rf values of 0.80-0.85 and 0.70-0.75 respectively,⁹⁹Tc^mthe Rf value of the colloid is 0.00-0.10; two kinds of reduction free in silica gel plate chromatography⁹⁹Tc^mHas Rf values of 0.95-1.00 and 0.80-0.85,⁹⁹Tc^mthe colloid has an Rf value of 0.00 to 0.10.

11. The use of reduced free technetium-99 m in the preparation of a diagnostic agent for tumor radioimaging.

12. Use of reduced free technetium-99 m in the preparation of a radioimaging therapeutic agent for tumors.

13. Use of reduced free rhenium-188 in the preparation of a tumour radioimaging therapeutic agent.

14. Use of reduced free technetium-99 m in preparing radioactive imaging diagnostic agent for ischemic and hypoxic diseases.

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