CN108434468B - A radioactive iodine-labeled protein binding ligand and its application - Google Patents

Abstract

The invention discloses a protein binding ligand labeled by radioactive iodine and application thereof, wherein the structural formula is as follows:

the I is radioactive iodine nuclide, R is OH or derived from PEG, folic acid, RGD, octreotide, epidermal growth factor, protein, nucleic acid or polysaccharide. The radiolabeled protein binding ligand labeling method provided by the invention is simple, low in cost and good in stability. The results of animal in-vivo experiments show that the albumin-bound ligand has higher uptake and longer retention time in blood, and has higher target/non-target ratio. The modified compound is modified on a targeting group or other functional groups, so that the pharmacokinetic property of the compound can be obviously improved, and the blood half-life of the compound is prolonged, so that the compound is suitable for being used as a blood pool, lymph and tumor diagnosis and treatment reagent.

Description

A radioactive iodine-labeled protein binding ligand and its application

technical field

The invention belongs to the technical field of diagnostic reagents, and particularly relates to a radioactive iodine-labeled protein binding ligand and application thereof.

Background technique

Molecular imaging is known as the medical imaging of the 21st century because it can provide a strong guarantee for the early detection, early diagnosis and early treatment of diseases. On the one hand, the development of imaging equipment has promoted the realization of early diagnosis and treatment of diseases; on the other hand, the development of non-invasive probes with good targeting, high specificity and high sensitivity has become a key element of molecular imaging. Only by obtaining probes with good performance can we better diagnose and treat diseases and evaluate prognostic effects.

In recent years, diseases of the cardiovascular system and malignant tumors have seriously affected human health, ranking the top two in terms of morbidity and mortality in the world population. Early diagnosis and treatment are important and urgent for reducing the harm of these two types of diseases and reducing the social burden. For cardiovascular diseases, it is necessary to use corresponding imaging methods to effectively display blood vessels and combine immunohistochemical studies to explore the pathogenesis of the disease. Early diagnosis, early treatment and individualized comprehensive treatment of such diseases are the most effective measures to reduce mortality. One of the fundamental basis for the indications and plans of early and individualized treatment is the diagnosis results of medical imaging. Therefore, sensitive and accurate imaging diagnosis technology is the key to realize the early diagnosis, early treatment and individualized treatment of cardiovascular diseases and tumors. key technology. At present, the commonly used vascular imaging methods include ink perfusion method, immunohistochemical method, tannic acid mordant method, enzyme histochemical method and so on. However, these methods have their own drawbacks, for example, they cannot be combined with immunohistochemical studies, the process is cumbersome, or the fluorescence is easily quenched. Imaging of the cardiovascular system using radionuclide-labeled imaging agents is one of the hotspots in radiopharmaceutical research today. Radionuclide-labeled blood pool imaging agents can non-invasively measure local or overall cardiac function in vivo, provide certain cardiac function parameters, and provide early diagnosis, prognosis and curative effects for coronary heart disease, cardiomyopathy and valvular disease. Observations have some value. Most of the diagnostic and therapeutic drugs for cardiovascular diseases have the following problems: such as short blood half-life, fast renal clearance, and easy excretion from the body, which makes it necessary to use high doses or frequent dosing, etc., which will inevitably lead to It produces a series of toxic and side effects, thus limiting its use. Currently, the most widely used radiolabeled blood pool imaging agent is ^99m Tc-RBC (red blood cells) (Stedrova V, et al. Eur JNucl Med Mol I 2010, 37: S495-S495.); ¹¹ C or ¹⁵ O Labeled RBCs (Kearfott KJ. JNucl Med 1982, 23(11): 1031-1037.); ⁶⁸ Ga-labeled Evans Blue derivatives (Zhang JJ, et al. J Nucl Med 2015, 56(10): 1609-1614. )Wait.

In the diagnosis and treatment of tumors, rapid blood clearance is generally considered an advantage in receptor-targeted imaging of tumors. Rapid blood clearance reduces radiotoxicity in normal tissues and increases the probe's target/background ratio in vivo, however, the short blood half-life also results in a relatively short uptake and residence time of molecular probes in tumor tissues. And long blood half-life is very important for tumor radionuclide targeted therapy. Fortunately, there are currently some methods that can be used to prolong the blood half-life, such as increasing the size of the drug molecule, pegylation, glycation, microencapsulation, binding to albumin, etc. Longer circulation time in the blood is beneficial to the diagnosis and treatment of malignant tumors. In addition, slowing the release rate of the drug over time may also allow continued uptake of the targeted organ or tissue for therapeutic benefit.

Human serum albumin (HSA) is a natural component of human blood, a single-peptide chain macromolecular protein composed of 576 amino acids, including 56 lysine residues, 17 tyrosine phenolic hydroxyl groups and a free thiol group , with a molecular weight of 68400 and a long biological half-life. ^99m Tc-HSA can be used as a blood pool imaging agent. Generally, the labeling rate of direct labeling method is not high, and the stability of the label is poor, so that the clearance rate in blood is fast, and the imaging effect is poor. In recent years, there have been many reports on the use of bifunctional chelating agents coupled to human serum albumin for ^99m Tc labeling, including: DTPA (diethylenetriaminepentaacetic acid), DMP (2,3-dimercaptopropionic acid) and other bifunctional chelating agents . However, ^99m Tc-DTPA-HSA is rapidly cleared from the blood, and imaging is required 5 to 10 minutes after injection, and there is a certain concentration in the liver, which is not conducive to the diagnosis of some diseases. A relatively successful one is ^99m Tc-DMP-HSA (Cambier JP, eta1. NuclMed Commun 1997, 18: 31-37.), and the preparation method of this complex has been kitted. In addition to the above-mentioned ^99mTc -labeled imaging agents, there are also ^62Cu , ^67Cu and ^68Ga radionuclide-labeled HSA and its derivatives. Although the direct in vitro radionuclide labeling of albumin is a relatively mature method, it is a human extract, which is expensive, may cause immune rejection when injected into the body, and the protein is easily contaminated and easily denatured, all of which are to a certain extent. limit its application. It can be seen from this that the existing relatively successful serum albumin labeling methods still have disadvantages such as complex preparation methods, high prices, easy infection with viruses, and the imaging effect is easily affected by other factors. The low-cost, high-efficiency radiolabel that can effectively bind to albumin will have broad application prospects.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a radioactive iodine-labeled protein binding ligand.

Another object of the present invention is to provide a method for preparing the above-mentioned radioiodine-labeled protein-binding ligand.

Another object of the present invention is to provide the application of the above-mentioned radioactive iodine-labeled protein-binding ligand.

The technical scheme of the present invention is as follows:

A radioactive iodine-labeled protein-binding ligand whose structural formula is as follows:

上述I为放射性碘核素，R为OH或衍生自PEG、叶酸、多肽、表皮生长因子、蛋白质、核酸或多糖的基团。

The above I is a radioactive iodine nuclide, and R is OH or a group derived from PEG, folic acid, polypeptide, epidermal growth factor, protein, nucleic acid or polysaccharide.

In a preferred embodiment of the present invention, the radioiodine is ¹³¹ I, ¹²⁵ I, ¹²⁴ I or ¹²³ I.

The preparation method of above-mentioned protein binding ligand, when R is OH, its synthetic route is as follows:

In a preferred embodiment of the present invention, the following steps are included:

(1) Dissolving 4-[(4-boronic acid phenyl) butyric acid 4-BBA in the reaction solvent, adding the same molar amounts of dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS respectively, The reaction was stirred at room temperature for 10-12 h, then by-products were removed by filtration, and the obtained filtrate was concentrated to obtain a white solid powder, namely 4-BBA-NHS, the activated ester of 4-[(4-boronylphenyl)butanoic acid;

(2) in the activated ester 4-BBA-NHS of 4-[(4-boronic acid group phenyl) butyric acid, add the catalyst that mixes cuprous oxide and 1,10-phenanthroline;

(3) adding the material obtained in the step (2) into the acetonitrile solution of the radioactive iodine nuclide, and shaking the reaction to obtain the labeled product 4-[I]IBA-NHS;

(4) Add Na ₂ CO ₃ or NaOH to hydrolyze it, and add hydrochloric acid solution for neutralization, and then the protein-binding ligand can be obtained.

Further preferably, the reaction solvent includes dimethylformamide, tetrahydrofuran and dimethylsulfoxide.

Another preparation method of the above-mentioned protein-binding ligand, when R is a group derived from PEG, folic acid, polypeptide, epidermal growth factor, protein, nucleic acid or polysaccharide, its synthetic route is as follows:

In a preferred embodiment of the present invention, the following steps are included:

(1) Dissolving 4-[(4-boronic acid phenyl) butyric acid 4-BBA in the reaction solvent, adding the same molar amounts of dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS respectively, The reaction was stirred overnight. The by-products were removed by filtration, and the filtrate was concentrated to obtain a white solid powder, namely the activated ester 4-BBA-NHS of 4-[(4-boronylphenyl)butanoic acid;

(2) in the activated ester 4-BBA-NHS of 4-[(4-boronic acid group phenyl) butyric acid, add the catalyst that mixes cuprous oxide and 1,10-phenanthroline;

(3) adding the material obtained in the step (2) into the acetonitrile solution of the radioactive iodine nuclide, and shaking the reaction to obtain the labeled product 4-[I]IBA-NHS;

(4) React 4-[I]IBA-NHS and molecule R with amino structure in dimethylformamide, dimethyl sulfoxide or water at 20-70℃ for 30-60min, while adding a small amount of triethylamine and pyridine Or N,N-diisopropylethylamine to promote the reaction, after the reaction is completed, the solvent is blown dry to remove, and the protein-binding ligand is obtained after purification.

Further preferably, the reaction solvent includes dimethylformamide, tetrahydrofuran and dimethylsulfoxide.

The application of the above-mentioned protein-binding ligand in the preparation of diagnostic reagents.

The beneficial effects of the present invention are as follows: the radiolabeled protein binding ligand labeling method provided by the present invention is simple, low in cost and good in stability. The results of animal in vivo experiments show that the albumin-binding ligand has higher uptake and longer retention in the blood, and has a higher target/non-target ratio. Modifying it to targeting groups or other functional groups can significantly improve the pharmacokinetic properties of the compound and prolong its blood half-life, making it suitable for use as blood pool, lymph and tumor diagnosis and treatment reagents.

Description of drawings

Fig. 1 is a TLC analysis diagram of 4-[ ¹³¹ I]IBA-NHS in physiological saline and acetonitrile within 24 hours respectively in Example 2 of the present invention.

Fig. 2 is the HPLC analysis chart of 4-[ ¹³¹ I]IBA and 4- ¹³¹ IBA-NHS in Example 2 of the present invention.

3 is a graph showing the results of dialysis experiments of 4-[ ¹³¹ I]IBA in the presence of human serum albumin in Example 2 of the present invention, respectively.

Fig. 4 is the comparison diagram of the dialysis experiment results of 4-[ ¹³¹ I]IBA and 4-[ ¹³¹ I]IBA-PEG in the presence or absence of bovine serum albumin respectively in Example 2 of the present invention;

5 is a graph showing the results of biodistribution of 4-[ ¹³¹ I]IBA, 4-[ ¹³¹ I]IBA-PEG and 4-[ ¹³¹ I]IBA-FA in mice respectively in Example 2 of the present invention;

Figure 6 is the cytotoxic MTT experiment after co-incubation of precursors with different concentrations and LO2 normal stem cells for 10 and 48 h in Example 2 of the present invention;

Figure 7 is the effect of different amounts of labeled precursors on the binding of 4-IBA to albumin in Example 2 of the present invention;

Figure 8 is a Micro SPECT/CT image of the protein-binding ligand in Example 2 of the present invention;

Figure 9(a) shows the change trend of tumor volume in mice treated with different treatment regimens; (b) shows the change trend of mouse body weight during treatment:

Figure 10(a) is a graph of IC ₅₀ of 4-[ ¹³¹ I]IBA; (b) is a graph of IC ₅₀ of radioiodinated Evans blue.

Detailed ways

The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.

Example 1

1.4-[ ¹³¹ I]IBA-NHS synthesis

Dissolve 1g of 4-[(4-boronylphenyl)butyric acid (4-BBA) in 15mL of DMF, add 981mg of dicyclohexylcarbodiimide and 548mg of N-hydroxysuccinimide respectively, and stir the reaction at room temperature overnight . The by-products were removed by filtration, and the filtrate was concentrated to obtain a white solid powder, namely the activated ester of 4-[(4-boronylphenyl)butanoic acid;

Take 1 mg of the activated ester of 4-[(4-boronic acid phenyl) butyric acid (4-BBA-NHS), add 50 μL of the catalyst of cuprous oxide and 1,10-phenanthroline mixed uniformly to it; add the above solution to Put it into the dried radioactive iodine, shake the reaction at room temperature for 30min, and then the labeled product 4-[ ¹³¹ I]IBA-NHS can be obtained; add an appropriate amount of Na ₂ CO ₃ to hydrolyze it to obtain the target product 4-[ ¹³¹ I ]IBA. The labeled final product was identified by HPLC, and its radioactive peak time matched that of the stable albumin ligand 4-[ ¹²⁷ I]IBA (the UV peak time of the stable ligand 4-[ ¹²⁷ I]IBA). was 3.92 min, and the radioactive peak time of 4-[ ¹³¹ I]IBA was 4.16 min).

2.4-[ ¹³¹ I]IBA-PEG synthesis

4-[ ¹³¹ I]IBA-NHS and NH2-PEG ₅₀₀₀ -N3 were reacted in DMF at room temperature for 1 h, and 10 μL of N,N-diisopropylethylamine was added to promote the reaction, and the target compound 4-[ ¹³¹ was finally obtained. I]IBA-PEG:.

3.4-[ ¹³¹ I]IBA-FA synthesis

4-[ ¹³¹ I]IBA-NHS and FA-PEG-NH ₂ were reacted in dimethylformamide (DMF) at room temperature for 1 h, and 10 μL of N,N-diisopropylethylamine was added to facilitate the reaction. After the completion of the reaction, purification was carried out by HPLC to finally obtain the target compound 4-[ ¹³¹ I]IBA-FA.

The stable reference compound 4-[ ¹²⁷ I]IBA used in the above synthesis and common reagents such as cuprous oxide, 1,10-phenanthroline, dimethylformamide, N,N-diisopropylethylamine, Na ₂ CO ₃ All are commercially available.

Example 2

The following is a description of the performance measurement of the labels 4-[ ¹³¹ I]IBA, 4-[ ¹³¹ I]IBA-PEG or 4-[ ¹³¹ I]IBA-FA synthesized by the method of Example 1 above:

1. Analysis and identification by TLC and HPLC

The TLC analysis system of 4-[ ¹³¹ I]IBA is as follows: the system of petroleum ether: ethyl acetate = 1: 1 was analyzed by silica gel aluminum plate at different time points by TLC; the system of normal saline was analyzed at different time points by using Polyamide films were analyzed by TLC. The labeling rate of 4-[ ¹³¹ I]IBA is fast, exceeding 99% within 10 min; the radiochemical purity is high, more than 95%; the property is stable and difficult to decompose, and it is still stable in normal saline and acetonitrile solvent until 24 hours. , TLC showed that only a single peak appeared, and the results are shown in Figure 1.

The HPLC analysis system of 4-[ ¹³¹ I]IBA was as follows: Perkin-Elmer Series 200LC equipped with Waters 2784 dual absorption wavelength UV detector and Bioscan radioactivity detector, Waters Symmetry C18 analytical column (5 μm, 150×3.9 mm). Flow rate 1 mL/min, elution gradient: 0-30 min: 80% methanol and 20% water, kept unchanged. The HPLC results are shown in Figure 2. The results show that the retention times of 4-[ ¹³¹ I]IBA-NHS and 4-[ ¹³¹ I]IBA are 5.23 min and 4.16 min, respectively, and the radiochemical purities are calculated to be greater than 95%.

2. Determination of fat-water distribution coefficient

The lipid-water distribution coefficient (log P) determination of ¹³¹ I-labeled albumin complex 4-[ ¹³¹ I]IBA and its derivatives was accomplished by the following steps:

Add 100 μL of the diluted radioactive injection to a centrifuge tube containing 2.9 mL of ultrapure water and 3 mL of n-octanol mixture (PBS and n-octanol were mixed the day before the experiment and left to stand for the next day when the experiment was performed. ), vortexed for 2 min, centrifuged at 6000 rpm for 5 min, took 100 μL of liquid from the aqueous phase and the n-octanol phase and counted by γ-counter radioactivity. The experiment was repeated three times and the average value was obtained. The formula for calculating the fat-water distribution coefficient (log P) is:

P=(I _a -I)/(I _b -I)

where _Ia represents the radioactive counts determined in the organic phase, _Ib represents the radioactive counts determined in the aqueous phase, and I represents the background counts.

Through calculation, the lipid-water distribution coefficient of each radiolabeled targeting probe was finally determined.

The results showed that the lipid-water distribution coefficient of 4-[ ¹³¹ I]IBA, logP=1.01±0.03, was lipid-soluble; the logP=-1.438±0.03 of 4-[ ¹³¹ I]IBA-PEG showed water-solubility. The water solubility of the PEG-modified labeled probe was significantly improved; the logP of 4-[ ¹³¹ I]IBA-FA was 0.52±0.03.

3. Determination of Albumin Binding Properties

The albumin binding capacity of 4-[ ¹³¹ I]IBA and 4-[ ¹³¹ I]IBA-PEG was determined by dialysis experiments, respectively. The molecular weight cut-off of the dialysis bag used was 8000-14000 Da, and the dialysate was 1000 mL of PBS 7.4 solution. Accurately weigh 30 mg of human serum albumin (HSA) or bovine serum albumin (BSA) and dissolve it in 3 mL of PBS7.4, add labeled 4-[ ¹³¹ I]IBA 18MBq, mix well and add it to the dialysis bag, then Placed in dialysate for dialysis. At different time points, 1 mL of dialysate was taken into centrifuge tubes, and immediately supplemented with 1 mL of PBS, and the radioactivity count of the dialysate was measured by γ-counter. At the same time, the radioactivity in the dialysis bag at different time points was measured with a radioactivity meter. As a control, take another tube of PBS7.4 without HSA or BSA, add the same dose of 4-[ ¹³¹ I]IBA, put it in a dialysis bag for dialysis, and measure its radioactivity in the same way. The above experiment was repeated three times to obtain the average value, and finally a concentration/radioactivity percentage curve was made, as shown in Figure 3. The results showed that in the presence of serum albumin, the radiolabel was not easily dialyzed out of the dialysis bag until At 24h, most of the radioactivity was still trapped in the dialysis bag, and the radioactive label in the dialysis bag without albumin was quickly removed, indicating that 4-[ ¹³¹ I]IBA can bind to serum albumin and is relatively stable. No decomposition occurred within 24h.

In order to compare the changes in the binding ability of 4-[ ¹³¹ I]IBA to albumin after PEG modification, the same method as above was used to test in bovine serum albumin (BSA) ^. I] Compared with IBA, after PEG modification, more radioactivity was trapped in the dialysis bag in the presence of bovine serum albumin, while the radioactivity in the dialysis bag without bovine serum albumin was quickly removed, indicating that PEG The modified 4-[ ¹³¹ I]IBA-PEG can also effectively bind to albumin.

4. Biodistribution Experiment

18-20g female BALB/c mice were used in the experiment. There were 3 mice in each group, each mouse was injected with the radioactive tracer 370KBq through the tail vein, and then sacrificed at different time points, dissected, collected blood and its organs, measured the weight of blood and organs, and was treated with γ- The counter measures the radioactivity count, and calculates the amount of radioactivity contained in the unit mass organ at different time points, which is expressed by %ID/g. The results are shown in Figure 5. The results showed that the amount of radioactivity in the blood was 10.51±2.58% ID/g 30 min after the injection of the radiolabeled 4-[ ¹³¹ I]IBA. The major organs such as liver, lung and kidney had the highest radioactive content at 2.93±0.11%ID/g, 4.47±0.13%ID/g and 5.68±0.40%ID/g, respectively. . Moreover, the thyroid gland, as the most sensitive gland to iodine ion, has very low uptake in each time period, indicating that the tracer is very stable in the body, and no deiodination occurs. The ratio of radioactivity in blood/organ was also the highest in each observation time period, reaching a higher target/non-target ratio. The experimental results show that the tracer 4-[ ¹³¹ I]IBA can effectively prolong the blood half-life and reduce the renal clearance rate. It is an ideal blood pool imaging agent.

In order to better improve the pharmacokinetic properties of 4-[ ¹³¹ I]IBA, PEG was introduced to modify it, in order to enhance its water solubility and further prolong the blood half-life. 4-[ ¹³¹ I]IBA-PEG was injected into mice through tail vein for biodistribution experiments. After 30 minutes, the radioactive content in blood was 24.79±0.89%ID/g, much higher than other tissues and organs. After longer time points such as 24h, the blood still contained higher radioactivity retention.

After the introduction of the folic acid targeting group, the circulation time of the probe in the blood was maintained. Since the kidney tissue highly expresses the folate receptor, the uptake of the probe in the kidney is high, further indicating the targeting of the folate group. In other organs such as liver and lung, radioactive retention is lower, which is more favorable for tumor imaging.

5. Cytotoxic MTT Assay

Normal hepatocytes LO2 were selected in the experiment, and the log-phase cells were collected after incubation, and the concentration of the cell suspension was adjusted. After the cells adhered, the prodrugs of 4-IBA with different concentration gradients were added in sequence, and 5 duplicate wells were set up to incubate at 37 °C for 10 h. After 24 h, 10 μL of 5 mg/mL MTT solution was added, and the culture was terminated after culturing for 4 h. The culture medium in the well was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. After dissolving the formazan in the cells, the absorbance value was detected by an enzyme-linked immunosorbent assay at a wavelength of 490 am, which can indirectly reflect the number of living cells. The results are shown in Figure 6, the addition of different concentrations of prodrugs did not have a significant effect on cell viability.

6. Effects of different concentrations of precursors on the binding of 4-IBA to proteins

Take about 5 μCi of radiolabeled tracer 4-[ ¹³¹ I]IBA, mix it with 2 mg/mL human serum albumin, add 4-IBA precursors with different concentration gradients and incubate for 30 to 60 min, and then place it in the Centrifuge in a 10kDa ultrafiltration centrifuge tube, set the centrifuge speed at 10000r/min, centrifuge for 10min, collect the liquids in the upper and lower layers of the centrifuge tube respectively, and measure the radioactivity count in a γ-counter detector. It can be seen from Fig. 7 that different concentrations of precursors were respectively added to the reaction system in which 4-[ ¹³¹ I]IBA was bound to albumin, and the binding amount of 4-[ ¹³¹ I]IBA to albumin was not affected.

7. MicroSPECT imaging in mice

The compound 4-[ ¹³¹ I]IBA-PEG with a radiochemical purity greater than 95% was prepared according to the examples, and 0.1 mL (about 3.7 MBq) was injected into healthy female BALB/c mice (about 18-20 m in weight) through the tail vein. g), and static SPECT/CT image acquisition was performed immediately. They were scanned at different time points after the injection of the radioactive tracer, and the imaging results are shown in Figure 8. 4-[ ¹³¹ I]IBA-PEG could be retained in the blood vessels and heart of mice, and extended to 4 h over time A clearer image in the blood pool can still be seen, indicating that the structure has a long blood half-life in vivo, which provides the possibility for the diagnosis, curative effect and prognosis evaluation of cardiovascular system diseases.

8. Radionuclide therapy experiments

The tumor therapeutic effect of 4-[ ¹³¹ I]IBA was investigated. 4T1 tumor cells were subcutaneously inoculated into the right hind leg of female BALB/c mice (weight about 18-20 g), and the treatment was started after the tumor grew to about 0.5 cm in diameter one week later. 15MBq 4-[ ¹³¹ I]IBA was injected into the mice through the tail vein, the tumor size of the mice was measured by vernier calipers every other day, and the weight changes of the mice were also measured. Tumor volume V=a×b ² /2. In the double-course treatment group, 15MBq 4-[ ¹³¹ I]IBA was injected again on day 5 after the first injection of 15MBq 4-[ ¹³¹ I]IBA. As controls, physiological saline and Na ¹³¹ I control groups were set, and 200 μL of physiological saline and 15MBq Na ¹³¹ I solution were injected respectively. The treatment results are shown in Figure 9. The tumor growth trend in the ^4-131 IBA treatment group was significantly slower. The tumor growth inhibitory effect was more obvious in the double-course group. The tumor growth rate in the normal saline group and Na ¹³¹ I group was significantly higher than that in the treatment group. After the injection of the radionuclide, the weight of the mice decreased slightly and returned to normal quickly, and no mice died during the treatment.

4-[ ¹³¹ I]IBA is a new radiolabeled albumin complex. Compared with the existing commonly used blood pool imaging agents, its radioactive ray penetration is strong, and it can perform non-invasive blood pool imaging in vivo. The obvious difference from the existing radioactive blood pool imaging agents ^99m Tc-RBC and ^99m Tc-HSA is that the latter two are biological extracts, the labeling process is complicated, the labeling product is unstable, and multi-step purification is required after synthesis. This also inevitably leads to the loss of products and radioactivity; and the protein is easily denatured, and may also produce immune rejection, which is expensive, and is also susceptible to virus infection. Radionuclide-labeled Evans blue has been widely studied as a better blood pool imaging agent (CN201310157168.9). The IC ₅₀ values of 4-[ ¹³¹ I]IBA and radioiodine-labeled Evans blue were determined by the competitive binding method to be 46.5 μM and 25.1 μM, respectively (Fig. 10). Evans blue showed higher affinity for proteins . However, in tumor and blood pool imaging, very high affinity is not the best choice. We hope to find a dynamic balance. After the imaging agent is combined with the protein, it can achieve a higher uptake at the lesion site, and hope to stay in the tumor. The radioactivity in normal tissue and blood is cleared faster, which not only increases the contrast between target and non-target, but also reduces the damage of radiopharmaceutical to normal tissue. Therefore, the level of affinity is not the most important indicator for evaluating the quality of the imaging agent, and its advantages and disadvantages should be comprehensively considered in combination with other factors. A series of experiments proved that the binding ability of 4-[ ¹³¹ I]IBA to protein is enough to make it used as an imaging agent for blood pool, lymph and tumor. Furthermore, the preparation process of Evans blue label requires relatively harsh conditions and tedious steps: 1) It needs to be chemically modified by a bifunctional chelator (NOTA or DOTA) to label the nuclide; 2) The positron used Most of the nuclides need an accelerator for preparation, which is cumbersome to operate and has a high operating cost; 3) the half-life of nuclides is short and cannot be transported long-distance; 4) After labeling, it needs to be purified by high performance liquid chromatography to meet the application requirements. In contrast, radioactive iodine-labeled 4-IBA has the advantages that radioactively labeled Evans blue probes do not have: 1) the reaction is simple and fast, and the labeling can be completed in a short time with high yield; 2) the labeling rate High, no purification is required, which can meet the needs of large-scale preparation in hospitals, and the excess labeling precursor has low biological toxicity and will not affect the binding of 4-IBA to albumin; 3) In the molecular structure, it can be further processed by chemical reactions Modified to obtain radiotracers or therapeutic drugs with better performance, without the need to introduce additional bifunctional chelators; 4) Radioactive iodine has multiple isotopes, ¹³¹ I and ¹²⁴ I can be used as imaging nuclides for SPECT and PET respectively, ¹³¹ I and ¹²⁵ I can be used as radiotherapy nuclides to meet different needs. In general, 4-[ ¹³¹ I]IBA has the advantages that general blood pool imaging agents do not have: the compound is a small organic molecule with simple molecular structure, mild labeling conditions, and can react at room temperature, and the labeling method is simple and easy It has the advantages of high labeling rate and easy to obtain complexes with high radiochemical purity, long storage time, good stability, effectively prolonging the blood half-life and reducing the renal clearance rate.

The radionuclide used in the present invention may also include other radioactive iodine isotopes, such as ¹²⁵ I, ¹²⁴ I, etc., and the preparation method is similar.

The above are only the preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the description should still be covered by the present invention. In the range.

Claims (8)

1. A radioiodinated protein-binding ligand, characterized in that: the structural formula is as follows:

the above I is radioactive iodine, R is OH or a group derived from PEG or folic acid; the radioactive iodine is¹³¹I、¹²⁵I、¹²⁴I or¹²³I；

When R is a group derived from PEG or folic acid, the synthetic route is as follows:

2. a method of preparing a protein binding ligand according to claim 1, wherein: when R is OH, the synthetic route is as follows:

3. the method of claim 2, wherein: the method comprises the following steps:

(1) dissolving 4- [ (4-boranophenyl) butyric acid 4-BBA in a reaction solvent, respectively adding dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS with the same molar weight, stirring at room temperature for reaction for 10-12 h, then filtering to remove byproducts, and concentrating the obtained filtrate to obtain white solid powder, namely activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(2) adding a catalyst consisting of cuprous oxide and 1, 10-phenanthroline which are uniformly mixed into activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(3) adding the material obtained in the step (2) into acetonitrile solution of radioactive iodine, and carrying out oscillation reaction to obtain a labeled product 4- [ I ] IBA-NHS;

(4) adding Na₂CO₃Or hydrolyzing the protein by NaOH, and adding hydrochloric acid solution for neutralization to obtain the protein binding ligand.

4. The method of claim 3, wherein: the reaction solvent is dimethylformamide, tetrahydrofuran or dimethyl sulfoxide.

5. A method of preparing a protein binding ligand according to claim 1, wherein: when R is a group derived from PEG or folic acid, the synthetic route is as follows:

6. the method of claim 5, wherein: the method comprises the following steps:

(1) dissolving 4- [ (4-boranophenyl) butyric acid 4-BBA in a reaction solvent, respectively adding dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS with the same molar weight, and stirring for reacting overnight; filtering to remove by-products, and concentrating the filtrate to obtain white solid powder, namely the activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(2) adding a catalyst consisting of cuprous oxide and 1, 10-phenanthroline which are uniformly mixed into activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(3) adding the material obtained in the step (2) into acetonitrile solution of radioactive iodine, and carrying out oscillation reaction to obtain a labeled product 4- [ I ] IBA-NHS;

(4) reacting 4- [ I ] IBA-NHS and a molecule R with an amino structure in dimethylformamide, dimethyl sulfoxide or water at 20-70 ℃ for 30-60min, simultaneously adding a small amount of triethylamine, pyridine or N, N-diisopropylethylamine to promote the reaction to occur, drying and removing a solvent after the reaction is finished, and purifying to obtain the protein binding ligand.

7. The method of claim 6, wherein: the reaction solvent is dimethylformamide, tetrahydrofuran or dimethyl sulfoxide.

8. Use of the protein binding ligand of claim 1 for the preparation of a diagnostic or therapeutic agent.

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