CN101538284B - A kind of preparation method and application of 99Tcm-EDTA-MTI complex - Google Patents

Abstract

The invention discloses a preparation method and application of a ⁹⁹ Tc ^m -EDTA-MTI complex, which comprises two steps: a. the synthesis of the ligand EDTA-MTI and b. the preparation of ^{the 99} Tc ^m -EDTA-MTI complex, ^{The 99} Tc ^m -EDTA-MTI complex is obtained. The complex has high radiochemical purity, good stability, simple preparation and low price, and has a high uptake value and good retention in tumors. Tumor/blood, tumor/ It has the advantage of good target/non-target ratio in muscle, and is a new type of tumor hypoxia imaging agent with popularization and application value.

Description

A kind of preparation method and application of 99Tcm-EDTA-MTI complex

Technical field

The invention relates to the technical field of ⁹⁹ Tc ^m directly labeled radiopharmaceutical chemistry and clinical nuclear medicine, in particular to a preparation method and application of a ⁹⁹ Tc ^m -EDTA-MTI complex.

Background technique

In the practice of radiopharmaceutical chemistry and clinical nuclear medicine technology, a large number of clinical experiments have shown that many diseases, such as tumors, cardiovascular and vascular diseases, have a common and significant biological feature, which is tissue hypoxia. Therefore, understanding tissue oxygen levels is of great significance for the diagnosis and treatment of these diseases. Hypoxia imaging agents can selectively stay in hypoxic tissues or cells, and nuclear medicine imaging can directly provide evidence of dysfunction in organs. At present, hypoxic imaging agents can be roughly divided into nitroimidazoles and non-nitroimidazoles. The binding mechanism of nitroimidazoles to hypoxic tissues is generally considered to be that the nitro group of the ligand generates free radical anions under the action of intracellular enzymes, and the intermediates will be further reduced in hypoxic cells and remain in the cells, while In normal cells, this intermediate is quickly oxidized and excreted. ⁹⁹ Tc ^m -labeled hypoxic tissue imaging agent is an important content of radiopharmaceutical chemistry research in the world. So far, ^{the 99} Tc ^m -labeled nitroimidazole tumor hypoxia imaging agents that have been studied more are:

⁹⁹ Tc ^m -N2IPA(Taiwei Chu, Rujun Li, Shaowen Hu et al. Preparation and biodistribution of ftechnetium-99m-labeled 1-(2-nitroimidazole-1-yl)-propanhydroxyiminoamide(N2IPA) as a tumor hypoxia marker[J]. Nucl Med Biol 2004, 31: 199-203);

⁹⁹ Tc ^m -DTPA-metronidazole (Chen Yue, Mo Tingshu, Kuang Anren, etc. Preparation of hypoxic tissue imaging agent ⁹⁹ Tc ^m -DTPA-metronidazole and experimental study on tumor-bearing animals. Chinese Journal of Nuclear Medicine, 2000, 20(2):78-81);

In addition to the above two types of tumor hypoxia imaging agents, there are ⁹⁹ Tc ^m -BMS181321, ⁹⁹ Tc ^m -RP435, ⁹⁹ Tc ^m -BMS194796, ⁹⁹ Tc ^m -MAG3-nitroimidazole, ⁹⁹ Tc ^m -N ₄ IPA, ⁹⁹ Tc ^m N(NIET) ₂ , ⁹⁹ Tc ^m -EC-metronidazole, ⁹⁹ Tc ^m -MNZCAA, ⁹⁹ Tc ^m -metronidazole xanthate, ⁹⁹ Tc ^m -MN-RPMA, ⁹⁹ Tc ^m - Tumor hypoxia imaging agents such as (CO) ₃ -metronidazole IDA have their own advantages, but generally have some shortcomings: either the tumor uptake value is low or the liver uptake value is high, which is There is still a certain gap between them and the ideal hypoxia imaging agent. Therefore, it is of great practical significance to further develop a new type of tumor hypoxia imaging agent that is convenient for clinical application. Metronidazole (abbreviated as MTI) is a nitroimidazole compound that easily accumulates in hypoxic tissues. How to couple the cheap and easy-to-obtain bifunctional linker EDTA with MTI to generate an MTI-containing diester, and prepare a ⁹⁹ Tc ^m -EDTA-MTI complex by radioactive ⁹⁹ Tc ^m labeling for tumor hypoxia imaging is currently an important issue. One of the important issues to be solved in this technical field.

Contents of the invention

The purpose of the present invention is to provide a preparation method of ⁹⁹ Tc ^m -EDTA-MTI complex, which has high radiochemical purity, good stability, low price and easy availability, and is widely used in the field of tumor hypoxia imaging.

In order to achieve the above object, the present invention adopts the following technical scheme: a preparation method of ⁹⁹ Tc ^m -EDTA-MTI complex, the preparation steps are as follows:

a. Synthesis of ligand EDTA-MTI:

A certain amount of EDTA and acetic anhydride were respectively added into the reaction flask, anhydrous pyridine was used as the solvent, the temperature was controlled at 65°C, the reaction was stirred for 26 hours, and then suction filtered. After washing the solid with anhydrous ether, it was recrystallized from DMF-Et ₂ O solution to obtain a white solid. Add an appropriate amount of white solid and metronidazole into the reaction flask, use anhydrous pyridine as the solvent, control the temperature at 70°C, stir for 24 hours and then evaporate to dryness. Anhydrous diethyl ether was added to the residue, and recrystallized with absolute ethanol after suction filtration to obtain a light yellow solid.

Concrete synthetic route is:

b. Preparation of ⁹⁹ Tc ^m -EDTA-MTI complex:

Add 1 mL of aqueous solution containing 10 mg of EDTA-MTI ligand and 0.2 mL of SnCl ₂ 2H ₂ O in HCl (the mass concentration of SnCl ₂ 2H ₂ O in 0.2 mol/L HCl is 2.0 g/L) into the reaction vessel in sequence , adjust the pH value of the solution to 6, then add 1mL ⁹⁹ Tc ^m O ₄ ^-eluent (3.7×10 ⁷ Bq/mL), and heat in a water bath at 100°C for 15 minutes to obtain the ⁹⁹ Tc ^m -EDTA- MTI.

The above-mentioned chemical synthesis reagents are all commercially available, widely available sources.

^{The 99} Tc ^m -EDTA-MTI synthesized by the above method is a new type of tumor hypoxia imaging agent. As a targeting group, the MTI-containing diester is generated by reacting with the bifunctional linker EDTA, and the -COOH in the EDTA-MTI molecule is used to complex with ⁹⁹ Tc ^m to generate a ⁹⁹ Tc ^m -EDTA-MTI complex. The results of biodistribution experiments in tumor-bearing mice showed that: ⁹⁹ Tc ^m -EDTA-MTI has higher uptake and better retention in tumors, good target/non-target ratios such as tumor/blood, tumor/muscle, etc., and can become a A novel tumor hypoxia imaging agent.

The biodistribution data of ⁹⁹ Tc ^m -EDTA-MTI, ⁹⁹ Tc ^m -DTPA-metronidazole and ⁹⁹ Tc ^m -N2IPA in tumor-bearing mice were compared, and the results are shown in Table 1.

Table 1 Comparison of biodistribution data of ⁹⁹ Tc ^m -N2IPA, ⁹⁹ Tc ^m -DTPA-metronidazole, ⁹⁹ Tc ^m -EDTA-MTI in tumor-bearing mice (%ID/g, 4h pi)

The above results show that ⁹⁹ Tc ^m -EDTA-MTI has the highest uptake value in tumors, which is obviously better than the other two complexes, and the tumor/muscle and tumor/blood ratios are also good. ⁹⁹ Tc ^m -EDTA-MTI is a new type of tumor The hypoxic imaging agent has better biodistribution performance and can be popularized and applied.

Experiments show that the properties of ⁹⁹ Tc ^m -EDTA-MTI are as follows:

1. Chromatographic identification of ⁹⁹ Tc ^m -EDTA-MTI complex:

Thin-layer chromatography (TLC) identification: polyamide film is used as support, acetonitrile is used as developer; filter paper is used as support, acetone is used as developer.

The chromatography result (Rf result) of each component of table 2

⁹⁹ Tc ^m O ₄ ^{- 99} Tc ^m O ₂ .nH ₂ O ^{99Tc m} -EDTA-MTI Polyamide-acetonitrile 0.3～0.5 0.1 0.7～1.0 Filter paper - acetone 1.0 0.1 0.1

The radiochemical purity of the marker was greater than 90% as determined by the above chromatographic identification.

High-performance liquid chromatography (HPLC) identification: Shimadzu SCL-10AVP high-pressure liquid chromatography, Kromasil 100-5C18 reverse-phase column (25cm×4.6mm), Packard liquid scintillation analyzer. Phase A is pure water, phase B is methanol, the gradient is 0.01-30min, phase B is 5-100%, the injection volume is 5μl, the flow rate is 1ml/min, and the retention time (Rt) of each component measured is: ⁹⁹ Tc ^m O ₄ ^- : 5.0 min, ⁹⁹ Tc ^m -EDTA-MTI: 3.5 min. The obtained chromatographic results show that there are radioactive main peaks, and the radiochemical purity of the ⁹⁹ Tc ^m -EDTA-MTI complex is greater than 90%.

2. Determination of lipid-water partition coefficient of ⁹⁹ Tc ^m -EDTA-MTI complex

Take 1.0mL of pH7.4 (0.025mol/L) phosphate buffer in a 10mL centrifuge test tube, add 1.0mL n-octanol and 0.01mL ⁹⁹ Tc ^m -EDTA-MTI complex solution in the centrifuge test tube, cover the stopper, fully Shake well and centrifuge for 5min (4000r/min). Then take out 0.1mL from organic phase and water phase respectively, measure the radioactive count of two phases, and calculate its fat-water partition coefficient P (the radioactivity of P=organic phase activity/water phase), record logP= -2.30, indicating that ⁹⁹ Tc ^m -EDTA-MTI is a hydrophilic substance.

3. In vitro stability determination of ⁹⁹ Tc ^m -EDTA-MTI complexes

The labeled ⁹⁹ Tc ^m -EDTA-MTI was placed at room temperature for different times (1, 2, 3, 4, 5, 6 hours) to measure its radiochemical purity. The experimental results showed that ⁹⁹ Tc ^m -EDTA-MTI was placed After 6 hours, the radiochemical purity was greater than 90%, which indicated that ⁹⁹ Tc ^m -EDTA-MTI had good stability in vitro at room temperature and was suitable for clinical application.

4. Determination of charge properties of ⁹⁹ Tc ^m -EDTA-MTI complex

Experimental method by electrophoresis. The selected component of the solution medium is PBS (pH=7.4) buffer solution. Cut the Xinhua No. 1 paper into a rectangle with a length of 15cm and a width of 0.8cm, draw a sample line in the center of the paper strip, and mark the positive and negative poles. Use a capillary to spot the sample to be tested on the spotting line. After spotting, dip the two ends of the electrophoresis strip into the buffer tanks of the two corresponding electrodes according to the marked positive and negative electrodes. The paper strip is suspended and the spotting line is in the middle. Cover the airtight cover, turn on the power, and adjust the voltage at 150V. Start timing when the electrophoresis strip is completely wet, and measure the radioactive distribution on the electrophoresis strip after 3-4 hours of electrification. The results show that more than 90% of the radioactive counts are concentrated on the positive electrode, indicating that ⁹⁹ Tc ^m -EDTA-MTI is a negatively charged substance.

5. Biodistribution experiment of ⁹⁹ Tc ^m -EDTA-MTI complex in tumor-bearing mouse model:

Inject 0.10mL ⁹⁹ Tc ^m -EDTA-MTI solution (about 7.4×10 ⁵ Bq) into the tail vein of Kunming mice (female, about 18-20 g) bearing the S-180 sarcoma model. 2h, 4h decapitation. The heart, liver, lung, kidney, brain, muscle, bone, blood, tumor and other relevant tissues and organs were collected, cleaned and weighed respectively, and their radioactive counts were measured on the FT-603 well-type gamma scintillation probe. The number of tumor-bearing mice in the time period was 3. The percent injected dose per gram (%ID/g) was calculated for each tissue. The results are shown in Table 3.

Table 3: Biodistribution of ⁹⁹ Tc ^m -EDTA-MTI in mice bearing S180 sarcoma (n=3) %ID/g

6. SPECT imaging experiment of ⁹⁹ Tc ^m -EDTA-MTI complex tumor-bearing mice

Three S180-bearing mice were injected with 0.1mL (0.3mCi) of ⁹⁹ Tc ^m -EDTA-MTI respectively, and their limbs were fixed in prone position for SPECT imaging 4 hours after injection (the imaging SPECT instrument was HAWKEYE VG8, using a matrix of 128×128 To scan, one frame, the scan time is 300s). The results of SPECT imaging showed that ⁹⁹ Tc ^m -EDTA-MTI was obviously concentrated in the tumor focus.

7. Abnormal toxicity test of ⁹⁹ Tc ^m -EDTA-MTI complex in animals

According to the requirements of abnormal toxicity inspection in the second appendix of the Chinese Pharmacopoeia in 2005, the animal abnormal toxicity experiment was carried out on the ⁹⁹ Tc ^m -EDTA-MTI labeling solution. Take 0.5 mL of the prepared ⁹⁹ Tc ^m -EDTA-MTI (total volume 3.5 mL, total activity 1 mCi) labeling solution and slowly inject it into 20 g Kunming mice through the tail vein, a total of 5 mice, according to the Pharmacopoeia Observation was prescribed for 48 hours, no abnormal reaction was found, and no one died. The dosage conversion (per kilogram of body weight) is equivalent to 357 times the maximum possible dosage of the human body (taking 50 kilograms as an example), indicating that ⁹⁹ Tc ^m -EDTA-MTI has low toxicity and can ensure safe clinical use.

Detailed ways:

Describe the present invention in detail below by embodiment:

A ⁹⁹ Tc ^m -EDTA-MTI complex:

a. Synthesis of ligand EDTA-MTI:

Step 1: Add 3.00g H ₄ EDTA, 5.6mL Ac ₂ O and 5mL anhydrous pyridine into a 100mL round-bottomed flask, control the temperature at 65°C, stir for 26h and then filter with suction. The solid was washed with anhydrous ether, and then recrystallized with a 7:3 DMF-Et ₂ O solution to obtain 2.10 g of a white solid with a yield of 80%. Its infrared spectrum data are: v(IR)/cm ^-1 : 2852 (CH ₂ ), 1808, 1752 (C=O), 1077 (COC). Its melting point is: mp190～194℃.

Step 2: Add 0.30 g of the white solid obtained in the previous step, 0.40 g of metronidazole and 10 mL of anhydrous pyridine into a 100 mL round bottom flask, react at a temperature of 70° C. for 24 h, and evaporate to dryness under reduced pressure. Anhydrous diethyl ether was added to the residue and suction filtered. Recrystallized with absolute ethanol to obtain 0.31 g of light yellow solid, yield: 44%. Its infrared spectrum data (IR)/cm ^-1 : 3400 (OH), 1746 (C=O), 1365, 1531 (NO ₂ ), 1189, 825 (COC). ESI-MS: 599.0 (M+H ⁺ ). Nuclear Magnetic Spectrum: ( ¹ H NMR, D ₂ O): δ7.82(s, 2H, C=CH), δ4.49(t, 4H, -CH ₂ -), δ4.35(t, 4H, - CH ₂ -), δ3.72(s, 4H, N-CH ₂ -COOR), δ3.45(s, 4H, N-CH ₂ -COOH), δ2.31(s, 6H, N=C-CH ₃ ).

b. Preparation of ⁹⁹ Tc ^m -EDTA-MTI complex

Add 1 mL of aqueous solution containing 10 mg of EDTA-MTI ligand and 0.2 mL of SnCl ₂ 2H ₂ O in HCl (the mass concentration of SnCl ₂ 2H ₂ O in 0.2 mol/L HCl is 2.0 g/L) into the reaction vessel in sequence , adjust the pH of the solution to 6, then add 1 mL of ⁹⁹ Tc ^m O ₄ ^-eluent (3.7×10 ⁷ Bq/mL), and heat in a water bath at 100°C for 15 minutes to obtain ^{the 99} Tc ^m -EDTA-MTI.

Claims (2)

1. a preparation method ^{of99Tc m} -EDTA-MTI complex, its preparation steps are as follows: a. Synthesis of ligand EDTA-MTI: Add a certain amount of EDTA and acetic anhydride into the reaction flask, use anhydrous pyridine as solvent, control the temperature at 65°C, stir for 26 hours, then filter with suction, wash the solid with anhydrous ether, and recrystallize with DMF-Et ₂ O solution , to obtain a white solid; add an appropriate amount of white solid and metronidazole into the reaction flask, use anhydrous pyridine as a solvent, and control the temperature at 70°C, stir and react for 24 hours and then evaporate to dryness; add anhydrous ether to the residue, and after suction filtration Recrystallized with absolute ethanol to obtain a light yellow solid; Concrete synthetic route is: b. Preparation of ⁹⁹ Tc ^m -EDTA-MTI complex: Add 1 mL of aqueous solution containing 10 mg of EDTA-MTI ligand and 0.2 mL of 0.2 mol/L HCl solution containing 0.4 mg of SnCl ₂ 2H ₂ O to the reaction vessel in sequence, adjust the pH of the solution to 6, and then add 1 mL of radioactive concentration 3.7×10 ⁷ Bq/mL of ⁹⁹ Tc ^m O ₄ ^-eluent , heated in a water bath at 100°C for 15 minutes, ^{the 99} Tc ^m -EDTA-MTI complex was obtained. 2. The use of the ⁹⁹ Tc ^m -EDTA-MTI complex in claim 1 in the preparation of tumor hypoxia imaging agents.