CN110743017B

CN110743017B - Radiopharmaceutical targeting galectin-1 and preparation method thereof

Info

Publication number: CN110743017B
Application number: CN201911004744.XA
Authority: CN
Inventors: 刘昭飞; 王凡; 赖建豪; 卢德华
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2021-03-09
Anticipated expiration: 2039-10-22
Also published as: CN110743017A

Abstract

The invention discloses a targeted galectin-1 radiopharmaceutical and a preparation method thereof, the radiopharmaceutical is a conjugate of a radionuclide, a bifunctional chelating agent and PEG4-TDGd, and the radionuclide is labeled with PEG4-TDGd through the bifunctional chelating agent. The radioactive drug delivers the radioactive nuclide to the surface of tumor cells or tumor hypoxia parts of high expression galectin-1 in vivo through the specific recognition of TDGd to galectin-1 and the modification of PEG4 to pharmacokinetics, thereby realizing the specific nuclear medicine positron emission computed tomography of tumors and tumor hypoxia areas and the monitoring of the curative effect of interventional therapy means such as tumor radiotherapy and the like.

Description

Radiopharmaceutical targeting galectin-1 and preparation method thereof

Technical Field

The invention relates to the technical field of radiopharmaceuticals for imaging and diagnosing diseases, in particular to a novel micromolecular radiopharmaceutical for nuclear medicine Positron Emission Tomography (PET) imaging and diagnosis of tumor hypoxia and a preparation method thereof.

Background

Radiotherapy is an important treatment means for various malignant tumors clinically at present. Radiotherapy is commonly applied worldwide for the treatment of 50% to 60% of patients with tumors. Radiotherapy has good curative effect in early and middle stage tumors, but obvious tolerance phenomenon can be observed in stage IV tumors and the like. Radiotherapeutic tolerance impairs the effectiveness of radiotherapy in treatment, leading to a poor prognosis of treatment failure, recurrence, etc. There is now ample evidence that radiotherapy tolerance is closely related to tumor hypoxia. In hypoxic tumors, about three times the radiation dose is required to achieve the therapeutic effect of normal oxygen level tumor tissue. Therefore, the hypoxia level is an important index for predicting the curative effect of radiotherapy. In view of the key role of tumor hypoxia in radiotherapy tolerance, if tumor hypoxia can be monitored accurately, non-invasively, quantitatively and dynamically, an effective means is provided for early warning of tumor radiotherapy tolerance or tumor recurrence after radiotherapy and making or modifying a personalized treatment scheme of a patient in a targeted manner. Thereby making it possible to prolong the survival of patients and improve prognosis.

Galectins are members of a large family of Carbohydrate-binding lectins, characterized by tight binding to beta-galactosides via a conserved Carbohydrate Recognition Domain (CRD). In humans, the galectin family includes mainly galectin-1, -2, -3, -4, -7, -8, -9, -10, -12, -13, etc. subtypes. Wherein, the galectin-1 is a 14.5kD secretory protein, which shows high expression in various tumors such as breast cancer, lung cancer, colorectal cancer, glioma and the like, and is involved in malignant processes of various tumors such as angiogenesis, immunosuppression, tumor metastasis and the like.

Tumor hypoxia induces galectin-1 secretion, and elevated galectin-1 expression levels are found in various tumors (rectal cancer, prostate cancer, Kaposi's sarcoma, acute myelocytic leukemia cells, etc.) under hypoxic conditions. Galectin-1 expression in hypoxic tumor cells is primarily regulated by HIF-1 α, a regulation dependent on the action of Hypoxia Response Elements (HREs) located 441-423bp upstream of the transcription start site of Lgals1 gene. The design of a specific targeted small molecule radiopharmaceutical aiming at the galectin-1 highly expressed in the hypoxic region plays an important role in early imaging diagnosis of tumor hypoxia, guidance and monitoring of the curative effect of tumor radiotherapy and the like.

Disclosure of Invention

The invention aims to provide a radiopharmaceutical for galectin-1 specific nuclear medicine imaging and a preparation method thereof, and the radiopharmaceutical has low preparation cost, good stability in vivo and good tumor imaging and tumor hypoxia imaging effects.

The purpose of the invention is realized by the following technical scheme:

a radiopharmaceutical targeting galectin-1, which is a conjugate of a radionuclide, a bifunctional chelator and PEG4-TDGd, wherein the radionuclide is labeled with PEG4-TDGd through the bifunctional chelator; the PEG4-TDGd has a structure shown in a formula (1):

the PEG4-TDGd is PEG4 modified TDGd which is a Thiodigalactoside (TDG) derivative, the structural formula of the TDGd is shown in a formula (2), and the PEG4 is polyethylene glycol with the polymerization degree of 4. For TDGd, a triazole benzene ring modified at the C3 position is used for enhancing the binding specificity of galectin-1, and an azide modified at the C3' position is used for performing cycloaddition reaction with alkynyl to obtain triazole for connecting PEG 4. TDGd is a molecule that achieves galectin-1 targeting.

Further, the bifunctional chelating agent is any one of DOTA, NOTA or derivatives of both.

Further, the radionuclide is⁶⁸Ga、⁶⁴Cu or¹⁸F。

Further, the radioactive drug is a colorless transparent liquid injection.

The preparation method of the radiopharmaceutical for targeting galectin-1 comprises the following steps:

a. preparation of PEG 4-TDGd: dissolving TDGd in PBS buffer solution, adding alkynyl-modified PEG4(Alkyne-PEG 4-NH)₂) Adding a monovalent copper ion catalyst, uniformly mixing, reacting at room temperature for 3-5 h, filtering the reaction mixed solution, separating and purifying by semi-preparative HPLC, collecting a product peak product, and freeze-drying to obtain white powder, namely NH₂-PEG4-TDGd；

b. Preparation of bifunctional chelating agent-PEG 4-TDGd: NH (NH)₂Dissolving PEG4-TDGd in an alkaline buffer solution, adding a bifunctional chelating agent DOTA or NOTA, and mixingAfter being mixed, the mixture reacts for 5 hours at room temperature, the reaction mixture is separated and purified by semi-preparative HPLC, the peak product of the product is collected and lyophilized to obtain white powder, namely DOTA-PEG4-TDGd or NOTA-PEG 4-TDGd;

c. preparation of radionuclide-bifunctional chelating agent-PEG 4-TDGd: dissolving DOTA-PEG4-TDGd or NOTA-PEG4-TDGd obtained in the step b into weak acid buffer solution, and adding radionuclide⁶⁸Ga、⁶⁴Cu or¹⁸F, wherein¹⁸F-labeling only reacted with NOTA-PEG4-TDGd and AlCl was added₃Heating in water bath at 80-120 deg.C for 10-20 min to obtain the final product⁶⁸Ga/⁶⁴Cu/¹⁸F-NOTA-PEG4-TDGd or⁶⁸Ga/⁶⁴Cu-DOTA-PEG4-TDGd。

The medicine of the invention is prepared from galactose agglutinin-1 targeted thiodigalactoside derivative (PEG4-TDGd) connected with PEG4, bifunctional chelating agent (DOTA or NOTA) and radionuclide (B)⁶⁸Ga、⁶⁴Cu or¹⁸F) And (4) forming. Wherein TDGd is formed by connecting a triazole benzene ring at the C3 position on the basis of Thiogalactoside (TDG) to improve the targeting affinity to galectin-1, and the azido is added at the C3' position and is connected with Alkyne-PEG4-NH through cycloaddition reaction with alkynyl₂Synthesis of NH₂-PEG4-TDGd。NH₂Coupling of PEG4-TDGd with the bifunctional chelating agent DOTA, NOTA or derivatives thereof followed by radionuclide⁶⁸Ga、⁶⁴Cu or¹⁸F is marked on PEG4-TDGd, so that the radioactive nuclide is carried to the tumor cells or tumor hypoxia parts with high expression of the galectin-1 in vivo through the specific recognition of the TDGd and the galectin-1, and the tumor or tumor hypoxia is subjected to noninvasive imaging diagnosis and guided radiotherapy by utilizing nuclear medicine Positron Emission Tomography (PET).

The invention has the beneficial effects that:

1. according to the invention, through connecting and modifying TDGd, a conjugate of radionuclide, a bifunctional chelating agent and PEG4-TDGd is obtained, namely the targeted galectin-1 radiopharmaceutical. The medicine is a single compound component, and can be specifically combined with human galectin-1 and mouse galectin-1. The medicine can be rapidly removed from normal tissues (such as liver) and blood in vivo, and can realize high-contrast PET imaging of tumor cells or hypoxic region of tumor tissue.

2. The galectin-1 targeting TDGd is a sulfo-digalactoside structure modified by triazole at the C3 position, and the triazole benzene ring connected at the C3 position increases the affinity to galectin-1 and the in vivo stability, so that the better in vivo galectin-1 targeting is achieved. C3' is connected with azido group, and can generate cycloaddition reaction with alkynyl group to obtain triazole which is used for connecting PEG 4.

3. The number of TDGd connected with PEG is 4 (namely PEG4), thereby increasing the water solubility of TDG micromolecules, prolonging the blood circulation time and improving the metabolism condition in vivo.

4. By using the preparation method of the invention, the radioactive nuclide is processed by a bifunctional chelating agent (DOTA or NOTA)⁶⁸Ga、⁶⁴Cu or¹⁸And F, labeling the DNA to PEG4-TDGd, carrying the radionuclide to the tumor part through the specific identification of the TDGd on the galectin-1, and carrying out non-invasive real-time dynamic diagnostic imaging and tumor radiotherapy guidance on hypoxic oxygen caused by diseases such as tumors and the like by using non-invasive, high-sensitivity, high-specificity and accurate quantitative nuclear medicine Positron Emission Tomography (PET).

The invention is further explained with reference to the drawings and the embodiments.

Drawings

FIG. 1 shows the structure of radionuclide-NOTA-PEG 4-TDGd (wherein the radionuclide is⁶⁸Ga、⁶⁴Cu or¹⁸F)；

FIG. 2 shows the structure of radionuclide-DOTA-PEG 4-TDGd (where the radionuclide is⁶⁸Ga or⁶⁴Cu)；

FIG. 3,⁶⁸A synthetic route of Ga-NOTA-PEG 4-TDGd;

FIG. 4, synthetic route of TDGd;

FIG. 5,¹⁸A synthetic route of F-NOTA-PEG 4-TDGd;

FIG. 6,⁶⁸Ga-NOTA-PEG4-TDGd(⁶⁸Ga-NOTA-PTDGd) and (A) mouse galectin-1 (mGal-1) and(B) the binding specificity of human galectin-1 (hGal-1); a, P<0.05；**,P<0.01；

FIG. 7,⁶⁸Biodistribution of Ga-NOTA-PEG4-TDGd in sorafenib (sorafenib) induced 4T1 mouse breast cancer hypoxic model (n ═ 4); a, P<0.05；

FIG. 8,⁶⁸Ga-NOTA-PEG4-TDGd and hypoxia imaging agent¹⁸PET imaging comparison of F-FMISO in sorafenib-induced 4T1 mouse breast cancer hypoxic model (n-4); arrows indicate hypoxic tumor sites;

FIG. 9,⁶⁸PET imaging of Ga-NOTA-PEG4-TDGd in a mouse model of lotus 4T1 breast cancer predicts the curative effect of radiotherapy.

Detailed Description

The materials used in the examples of the present invention: Alkyne-PEG4-NH₂Purchased from sienna millennium biotechnology limited; 1,4,7, 10-tetraazadocaine-N, N' -tetraacetic acid-N-hydroxysuccinimide (DOTA-NHS) and S-2- (4-Isothiocyanatobenzyl) -1,4, 7-tetraazacyclonenane-1, 4, 7-tetraacetic acid (p-SCN-Bn-NOTA) are available from Macrocyclics, USA; Sep-Pak C18 column was purchased from Waters, USA. Radionuclides⁶⁴Cu is produced in nuclear medicine department of tumor hospital of Beijing university; radionuclides⁶⁸Ga is composed of⁶⁸Ge-⁶⁸Leaching the Ga generator to obtain; radionuclides¹⁸F is produced by a medical accelerator; other conventional chemicals were purchased from Sigma-Aldrich, usa; recombinant mouse and human galectin-1 were purchased from US R&D Systems, Inc.

Example 1:

this embodiment uses radionuclides⁶⁸Ga-labeled NOTA-PEG4-TDGd is exemplified.⁶⁸The Ga-NOTA-PEG4-TDGd radioactive drug is prepared from TDGd, polyethylene glycol PEG4, bifunctional chelating agent NOTA and radionuclide⁶⁸Ga. Wherein, the difunctional chelating agent NOTA can be replaced by DOTA,⁶⁸ga radionuclides may be exchanged for⁶⁴Cu or¹⁸F (shown in fig. 1 and 2). To be provided with⁶⁸Ga-NOTA-PEG4-TDGd is taken as an example, and the preparation method is shown in figure 3 and comprises the following steps:

a、NH₂preparation of PEG 4-TDGd: 6 mu mol of TDGd is dissolved in 500 mu L of PBS, and 36 mu mol of Alkyne-PEG4-NH is added₂0.6. mu. mol equivalent of CuSO₄And 3 mu mol of sodium ascorbate is mixed uniformly and then placed at room temperature for reaction for 3-5 h, and then the reaction mixed solution is filtered by a microporous filter membrane and injected into semi-preparative HPLC for separation and purification. The product peak was collected, rotary evaporated to remove acetonitrile and lyophilized to a white powder by a lyophilizer. Obtaining the product NH₂-PEG₄A total of 2.8mg of-TDGd. MALDI-TOF-MS mass spectrometry result is that m/z ═ 742.3[ MH]⁺(C₃₁H₄₇N₇O₁₂S theoretical value 741.8).

b. Preparation of bifunctional chelating agent-PEG 4-TDGd: 2. mu. mol of NH are taken₂-PEG₄TDGd dissolved in 500. mu.L of 0.1mol/L NaHCO₃To a buffer (pH 8.5-9.0) was added 6 μmol of p-SCN-Bn-NOTA (dissolved in DMF). After mixing uniformly, the mixture is placed at room temperature for reaction for 5 hours, and then the reaction mixture is injected into semi-preparative HPLC for separation and purification. The product peak was collected, rotary evaporated to remove acetonitrile and lyophilized to a white powder by a lyophilizer. The product NOTA-PEG4-TDGd was obtained in a total amount of 1.5 mg. MALDI-TOF-MS mass spectrometry analysis result shows that m/z is 1192.57[ MH []⁺(C₅₁H₇₃N₁₁O₁₈S₂Theoretical value 1192.3).

c、⁶⁸Preparation of Ga radionuclide-bifunctional chelating agent-PEG 4-TDGd: 20. mu.g of NOTA-PEG4-TDGd was dissolved in 500. mu.L of 0.1M NaOAc buffer (pH 5.5), to which 10mCi of⁶⁸GaCl₃Heating the leacheate in water bath at 100 ℃ for 15min to prepare the traditional Chinese medicine⁶⁸Ga-NOTA-PEG4-TDGd radiopharmaceutical.

The TDGd is prepared manually, and is not limited to a preparation method, and the preparation method adopted in this example is shown in fig. 4:

firstly synthesizing a compound 1, respectively converting the compound into two structural units of glycosyl halide (a compound 3) and triisopropylsilylthioglycoside (a compound 4), then substituting isomeric bromo of a compound 2 by the compound 4 after desiliconization and activation to obtain a compound 5, and then removing an acetyl protecting group and purifying to obtain a target product compound 6.

The method comprises the following steps:

a. n equivalents of compound 1 were weighed out and dissolved in acetonitrile, and 1.5n equivalents of ethynylbenzene, 1.5n equivalents of DIPEA, 1.5n equivalents of CuI were added. The reaction mixture was stirred at 30 ℃ for 16 h. Then concentrated by filtration under reduced pressure and purified by column chromatography. N equivalents of the intermediate product were dissolved in DCM solution and 14n equivalents of HBr were added. The mixture was stirred at 25 ℃ for 4 hours. Adding NaHCO₃The aqueous solution was bubbleless and extracted with DCM. The organic layer was concentrated under reduced pressure and purified by column chromatography to give compound 3.

b. Weighing n equivalents of compound 1 dissolved in DCM and EtOAc 1: 1 solution, 2n equivalents of TiBr are added₄. The reaction mixture was stirred at 30 ℃ for 12 h. Adding NaHCO₃The aqueous solution was bubbleless and extracted with DCM. The organic layer was concentrated under reduced pressure and subjected to column chromatography to give compound 2.

c. At n equivalents of compound 2CH₃CN solution, 3n equivalents of K₂CO₃And 1.5n equivalents of TIPSSH. The reaction mixture was stirred at 25 ℃ for 2 h. The reaction mixture was concentrated under reduced pressure and purified by column chromatography to give compound 4.

d. Dissolving n equivalents of compound 3 and n equivalents of compound 4 in CH respectively₃To CN, 1.5n equivalents of TBAF were added. The mixture was stirred at 30 ℃ for 0.1 h. The reaction mixture was then concentrated under reduced pressure and purified by column chromatography to give compound 5.

e. n equivalents of compound 5 are dissolved in methanol solution and 5.6n equivalents of NaOME are added. The mixture was stirred at 30 ℃ for 3 hours. Addition of H⁺The pH of the resin was adjusted to 7 using Prep-HPLC (mobile phase: [ water (0.04% NH) ]₃H₂O+10mM NH₄HCO₃)-ACN]And B%: 10 to 40 percent, 10.5min) to purify the reaction solution. The purified liquid was put into a freeze dryer for concentration and freeze-dried to obtain a white powder, to obtain the objective product (Compound 6).

Example 2:

this embodiment uses radionuclides¹⁸F-labeled NOTA-PEG4-TDGd is exemplified.¹⁸The F-NOTA-PEG4-TDGd radioactive drug is prepared from TDGd and polyethyleneDiol PEG4, bifunctional chelating agent NOTA and radionuclide¹⁸And F. Wherein¹⁸Radiolabelling of F with NOTA-PEG4-TDGd by AlCl₃The preparation method is shown in figure 5 and comprises the following steps:

a. NOTA-PEG4-TDGd was prepared as in example 1.

b、¹⁸Preparation of F radionuclide-bifunctional chelating agent-PEG 4-TDGd: 20. mu.g of NOTA-PEG4-TDGd was dissolved in a solution containing 2mM AlCl₃To 0.1M NaOAc buffer (pH 4.0), then 150 μ L of acetonitrile and 20mCi of water were added thereto¹⁸F, heating in a water bath at 110 ℃ for 15 min. After the reaction mixture was cooled to room temperature, it was diluted with 5mL of pure water and purified by Sep-Pak C18 column to obtain the product of the present invention¹⁸F-NOTA-PEG4-TDGd radiopharmaceutical.

Example 3:

this embodiment is as follows⁶⁸Ga-NOTA-PEG4-TDGd exemplifies the in vivo and in vitro binding specificity identification of galectin-1 targeted radiopharmaceuticals and PET imaging applications thereof. Wherein the content of the first and second substances,⁶⁸Ga-NOTA-PEG4-TDGd may also be⁶⁸Ga-DOTA-PEG4-TDGd、⁶⁴Cu-NOTA-PEG4-TDGd、⁶⁴Cu-DOTA-PEG4-TDGd or¹⁸F-NOTA-PEG4-TDGd。⁶⁸Binding specificity of Ga-NOTA-PEG4-TDGd to galectin-1:

coating the recombinant mouse galectin-1 or the human galectin-1 on a high adsorption ELISA plate, and then coating the recombinant mouse galectin-1 or the human galectin-1 on the high adsorption ELISA plate⁶⁸Ga-NOTA-PEG4-TDGd additions Radioactive binding experiments were performed in which the blocking group was added with excess unlabeled TDG to detect⁶⁸The binding specificity of Ga-NOTA-PEG4-TDGd to galectin-1. The experimental result shows that the material is sealed by TDG⁶⁸The binding of Ga-NOTA-PEG4-TDGd to mouse galectin-1 or human galectin-1 is remarkably reduced (see FIG. 6), which proves that the Ga-NOTA-PEG4-TDGd has specific binding to the mouse galectin-1 and the human galectin-1.

⁶⁸Biodistribution of Ga-NOTA-PEG4-TDGd in hypoxic mouse model:

BALB/c breast cancer mice bearing 4T1 mice were divided into 2 groups of 4 mice each. One group of themMice were gavaged with sorafenib (sorafenib) to induce tumor hypoxia, and another group of mice were given a blank vehicle control. Subsequent groups of mice were injected via tail vein⁶⁸Ga-NOTA-PEG4-TDGd, 1h after injection, mice were sacrificed, blood, tumors and major organs were removed, their radioactivity counts were weighed and measured, and the percent injected dose per gram of tissue (% ID/g) was calculated after decay correction. The results show that it is possible to display,⁶⁸uptake of Ga-NOTA-PEG4-TDGd was significantly higher in sorafenib-induced hypoxic tumors than control tumors (see figure 7). The experimental results show that the high-temperature-resistant steel,⁶⁸Ga-NOTA-PEG4-TDGd has specific high uptake in hypoxic tumors.

⁶⁸PET imaging of Ga-NOTA-PEG4-TDGd in hypoxic mouse tumor models:

in a sorafenib-induced hypoxic 4T 1-loaded breast cancer BALB/c mouse model, hypoxic imaging agents are respectively injected¹⁸F-FMISO and⁶⁸Ga-NOTA-PEG 4-TDGd. At the optimal imaging time after injection, PET images were scanned by a NanoScan PET/CT imaging instrument (Mediso, Hungary) and compared for tumor hypoxia imaging characteristics. As shown in fig. 8, compared with the current clinical anaerobic imaging agent¹⁸Compared with the F-FMISO method, the method has the advantages that,⁶⁸the Ga-NOTA-PEG4-TDGd has low background in the abdominal cavity and high contrast of hypoxic tumor imaging.

⁶⁸The Ga-NOTA-PEG4-TDGd mouse PET imaging predicts the curative effect of radiotherapy:

19 hypoxic-induced-sorafenib-induced, 4T 1-charged, breast cancer BALB/c mice were divided into 2 groups, a control group and a radiation-treated group, respectively. Among them, 6 mice were used as a blank control group and 13 mice were used as a radiation treatment group. On day 3, mice in the radiation treatment group were injected via tail vein⁶⁸Ga-NOTA-PEG4-TDGd, PET images were scanned by a NanoScan PET/CT imager 1h after injection, and tumor uptake was quantitatively analyzed. According to the quantitative⁶⁸SUV value of Ga-NOTA-PEG4-TDGd uptake in tumors is SUV>0.15 (high uptake) and SUV<Group 0.15 (low intake). Then, local radiotherapy was performed on the tumors of the radiotherapy group by an X-ray irradiator (RS2000 PRO, Radsource, usa) on

days

6, 8 and 10, respectively. Observation channel⁶⁸Ga-NOTA-PEG4-TDGd differentiated high tumor uptakeThe curative effect of tumor radiotherapy is obtained, wherein the curative effect of tumor radiotherapy is that the tumor radiotherapy has high hypoxic degree and low tumor uptake (the hypoxic degree is low). As can be seen from the experimental results,⁶⁸the tumor treatment effect of the Ga-NOTA-PEG4-TDGd low-uptake radiotherapy group is obviously higher than that of the Ga-NOTA-PEG4-TDGd low-uptake radiotherapy group⁶⁸The tumor treatment effect of the Ga-NOTA-PEG4-TDGd high-uptake radiotherapy group (see FIG. 9). The result indicates that⁶⁸Ga-NOTA-PEG4-TDGd noninvasive PET imaging evaluation of tumor hypoxia state, thereby early predicting the curative effect of radiotherapy.

In this embodiment, the galectin-1 targeting radiopharmaceutical can be used for hypoxia imaging of tumors and predicting the efficacy of tumor radiotherapy. The radiopharmaceutical of the invention specifically binds to the galectin-1 at the hypoxic part, so that the radiopharmaceutical can realize specific PET imaging diagnosis in other hypoxic-related diseases such as stroke and the like.

Claims

1. The application of a galactose lectin-1 targeted radiopharmaceutical in preparing a tumor hypoxia imaging drug is characterized in that the radiopharmaceutical is a conjugate of a radionuclide, a bifunctional chelating agent and PEG4-TDGd, and the radionuclide is labeled with PEG4-TDGd through the bifunctional chelating agent; the PEG4-TDGd has a structure shown in a formula (1):

wherein the radionuclide is⁶⁸Ga、⁶⁴Cu or¹⁸F; the bifunctional chelating agent is DOTA or NOTA.

2. The use of claim 1, wherein the galectin-1 targeting radiopharmaceutical is a colorless and transparent liquid injection.