CN100459932C - A kind of MRI molecular imaging probe and preparation method thereof - Google Patents

Abstract

The invention discloses an MRI molecular imaging probe and a preparation method thereof. The MRI molecular imaging probe provided by the present invention includes a central ion, a specific molecule and a chelating agent, the specific molecule is connected to the chelating agent, and the central ion and the chelating agent form a chelate, wherein the chelating agent includes a main Chelating agent and co-chelating agent, the main chelating agent is hydrazine hydrazine nicotinamide, and the co-chelating agent is selected from one of N-tris(hydroxymethyl)methylglycine and ethylenediaminediacetic acid. The present invention uses HYNIC and Tricine or EDDA as a chelating agent to synthesize an MRI molecular imaging probe, which has the following advantages: HYNIC has high specificity and can be combined with polypeptides; a small molecule chelating agent is used to eliminate the influence of chelating agent functional groups on the activity of polypeptides; HYNIC and Tricine and EDDA as chelating agents reduce the cost of probe synthesis; reduce the molecular weight of the probe to improve the penetration of the probe into the organism and improve the biological performance of the probe.

Description

A kind of MRI molecular imaging probe and preparation method thereof

technical field

The invention relates to an MRI molecular imaging probe and a synthesis and preparation method thereof.

Background technique

Magnetic resonance imaging (MRI) technology is one of the three major technologies of medical molecular imaging, and others include PET (SPECT) and optical imaging. Compared with PET technology, nuclear magnetic resonance imaging molecular imaging technology has the advantages of non-radioactive probes and high anatomical resolution of images; however, the disadvantages are low signal detection and specificity. At present, MR is still in the category of anatomical morphology diagnosis. Functional MR and MR spectroscopy can only be classified into the early stage of indirect molecular imaging. MR molecular imaging has the potential to become the most promising molecular imaging method. What is lacking now is the direct molecular imaging method. Targeted specific probes.

Probes are the basis of magnetic resonance molecular imaging (MRI). MRI molecular imaging probes include central ions that can be detected by MRI equipment, and specific molecules (including ligands, specific antibodies, etc.) that can specifically bind to targets. In order to make MRI molecular imaging probes highly stable and sufficient signal strength, this study developed and constructed MR targeting probes by itself, connecting specific molecules to chelating agents, and forming chelating complexes between central ions and chelating agents for targeted imaging of tumors or target lesions and Check out.

In MRI molecular imaging probes, the central ions are usually paramagnetic metal elements, and the commonly used paramagnetic metal central ions (paramagnetic metal ions) of transition metals and lanthanide metals include Fe ²⁺ , Fe ³⁺ , Mn ²⁺ , Gd ³⁺ and Dy ³⁺ etc. See Table 1 for a comparison of the binding properties of several paramagnetic metal ions to chelating agents (ligands).

Table 1 Comparison of the binding properties of paramagnetic metal ions and chelating agents (ligands)

paramagnetic metal ions Metal ion radius/nm A coordinating atom that easily binds to a metal ion A coordinating group that easily binds to metal ions Gd³⁺ 0.0938 Oxygen and Nitrogen Ligands Polyaminopolycarboxylates Fe²⁺, Fe³⁺ 0.076 Nitrogen ligand Carboxylic acid group, tyrosine, porphyrin Mn²⁺ 0.088 Oxygen and Nitrogen Ligands Carboxylate, Phosphate

These metal ions are all water-soluble. Gd ³⁺ is the most commonly used paramagnetic metal center ion. Gd ³⁺ has 7 unpaired electrons, large spin magnetic moment, symmetrical electric field, high relaxation efficiency, and easy coordination with water. , and the number of coordinated water molecules is 8 or 9, which is an ideal paramagnetic metal ion. However, free hydrated Gd ³⁺ and most complexes are not compatible with venous blood, and are easy to precipitate and have high toxicity. This requires the selection of a chelating agent that has very high stability after being combined with the metal ion.

The choice of chelating agent (complex) plays an important role in the preparation of MRI molecular probes. Chelating agents not only need to form highly stable chelates with paramagnetic metal ions, but also need to bind to specific molecules. Therefore, chelating agents play an important dual-functional role in MRI molecular probes.

Commonly used chelating agents are generally divided into two categories: linear and cyclic. No matter which kind of chelating agent is required to form a highly stable chelate with paramagnetic metal ions, and to be able to have a stable combination with specific molecules, it is also required that the formed chelate does not affect the biological performance of specific molecules. Since this type of chelating agent not only needs to link with the polypeptide, but also forms a chelate with the metal ion, it is also called a bifunctional conjugating agent (BFCA). For MRI-specific probes, both linear and circular chelators are bifunctional linkers.

At present, commonly used linear chelating agents include diethylenetriaminepentaacetic acid (DTPA) and its derivatives, etc., and circular chelating agents include 1,4,7,10-tetraazacyclododecane-1,4,7,10 -tetraacetic acid (DOTA), cyclic anhydrides DTPA and ethylenediaminetetraacetic acid (EDTA) and their derivatives, etc. The molecular formulas of DTPA and DOTA are shown in Formula I and Formula II respectively:

(Formula I) (Formula II)

In MRI molecular imaging probes, specific molecules include polypeptides that can specifically bind to enzymes and receptors; small molecules that can competitively bind to enzyme receptors; and antibodies that can specifically bind to antigens.

The research on the specific molecular selection of MRI probe binding target on the cell membrane surface shows that peptides and small molecules are better than antibodies. This is mainly due to the following problems in antibodies and specific monoclonal antibody probes: antibodies need to be desensitized when used; antibodies, including specific monoclonal antibodies, are still unstable in vivo, and it is difficult to meet expectations The purpose; the antibody has a large molecular weight and has poor penetration and permeability in the tissue, so the optimal imaging time is long. Generally, it takes about 24 hours, which is not conducive to clinical work; there are issues such as high environmental requirements for antibody labeling. Although the method of antibody fragments has been used for antibody imaging in recent years, it is still difficult to achieve the expected goal. For polypeptides and small molecule competitive inhibitors, no desensitization preparation is required, which greatly facilitates clinical work; the molecular weight of polypeptides is small, and their penetration and permeability in tissues are strong. The best imaging time is generally about 4 hours after injection. In this way, the inspection can be completed on the day when the molecular probe is injected; polypeptide molecules have high stability in vivo, and have minimal side effects on the human body in vivo; polypeptides and specific small molecules are easily connected with bifunctional chelating agents, making the The synthesis and preparation process is obviously simplified, which is convenient for research and clinical work; peptides have a high target-to-background tissue ratio due to their small molecules, so that the contrast of diseased tissue images can be significantly improved. Octreotide, an octapeptide analog of somatostatin, was the first to enter clinical application, which can specifically bind to somatostatin receptors (SSRT) and exert physiological effects. Table 2 is the specific MRI molecular probes linked by several different paramagnetic metal ion chelates.

Table 2 Specific MRI molecular probes linked by different paramagnetic metal ion chelates

From the point of view of current MRI molecular imaging probes, DTPA and DOTA chelating agents tend to be selected for connection with polypeptide molecules. However, DTPA and DOTA chelating agents have many difficulties in linking with polypeptides due to the following deficiencies:

1) DTPA and DOTA are multifunctional compounds. When connecting DTPA and DOTA chelating agents with polypeptides, it is necessary to protect the chelating agent and other functional groups of the polypeptide so as not to affect the functions of the polypeptide and chelating agent. After the synthesis is completed, the protective group on the functional group needs to be removed to restore the function of the chelating agent and the polypeptide. This obviously increases the complexity of synthesis and the cost of making the probe, making the MRI probe lose the value of actual clinical use.

2) Chelating agents with multifunctional groups will affect the biological activity of polypeptides.

3) The multifunctional chelating agent increases the volume of the probe.

Contents of the invention

The object of the present invention is to provide an MRI molecular imaging probe and a preparation method thereof.

The MRI molecular imaging probe provided by the present invention includes a central ion, a specific molecule and a chelating agent, the specific molecule is connected to the chelating agent, and the central ion and the chelating agent form a chelate, wherein the chelating agent includes a main Chelating agent and co-chelating agent, the main chelating agent is hydrazine hydrazine nicotinamide, and the co-chelating agent is selected from one of N-tris(hydroxymethyl)methylglycine and ethylenediaminediacetic acid.

In the MRI molecular imaging probe, when the secondary chelating agent is N-tris(hydroxymethyl)methylglycine, the main chelating agent hydrazinohydrazine nicotinamide can be combined with 2 molecules of N-tris(hydroxymethyl)methylglycine Glycine is used for chelation; when the auxiliary chelating agent is ethylenediamine diacetic acid, the main chelating agent hydrazinohydrazinonicotinamide can be chelated with 1 molecule of ethylenediamine diacetic acid.

In the present invention, the central ion is preferably Gd ³⁺ , Fe ²⁺ , Fe ³⁺ or Mn ²⁺ . The specific molecule is preferably octreotide and/or RGD peptide.

The preparation method of the MRI molecular imaging probe of the present invention comprises the following steps:

1) preparing a central ion solution;

2) Add N-tris(hydroxymethyl)methylglycine or ethylenediaminediacetic acid to the hydrazinohydrazine nicotinamide solution connected with specific molecules, and mix to obtain a chelating agent solution;

3) The central ion solution is added to the chelating agent solution, and the two solutions are mixed to obtain the MRI molecular imaging probe.

Wherein, in step 2), the molar ratio of hydrazinonicotinamide to N-tris(hydroxymethyl)methylglycine is 1:2, or the molar ratio to ethylenediaminediacetic acid is 1:1. But in the actual preparation process, the consumption of these two auxiliary chelating agents is often slightly excessive.

The present invention uses HYNIC and Tricine or EDDA as a chelating agent to chelate with Gd3 ⁺ (or Fe ²⁺ , Fe ³⁺ , Mn ²⁺ ) to synthesize an MRI molecular imaging probe, which has the following advantages:

HYNIC is highly specific and can bind to peptide molecules;

Small molecule chelating agents are used to eliminate the influence of chelating agent functional groups on the activity of polypeptides. Maintaining the correct spatial structure of polypeptides is an important prerequisite for exerting their physiological activities. Because macromolecular chelating agents contain many functional groups, they can change the spatial structure of polypeptides and affect their physiological activities. ; and the small molecule chelating agent has no redundant non-essential functional groups except the functional group connected to the end of the polypeptide, so it has a slight impact on the spatial structure of the polypeptide.

HYNIC, Tricine and EDDA are used as chelating agents to reduce the cost of probe synthesis;

Reducing the molecular weight of the probe improves the penetration of the probe into organisms and improves the biological performance of the probe. Studies have shown that when the molecular weight of the complex is above 50kd, the penetrability in vivo decreases significantly, and it is basically not suitable for specific imaging requirements; while the molecular weight of the probe of the present invention is below 2000, it still has a good use effect.

Description of drawings

Figure 1 is the T1WI images of the Gd-Tricine-HYNIC-Octreotide molecular probe in rabbits at different times.

Figure 2 is the T1WI images of the Gd-DTPA molecular probe in rabbits at different times.

Detailed ways

The molecular structures of hydrazinohydrazinonicotinamide (HYNIC), N-tris(hydroxymethyl)methylglycine (Tricine) or ethylenediaminediacetic acid (EDDA) are shown in formula III, formula IV and formula V respectively:

(Formula III) (Formula IV) (Formula V)

HYNIC can specifically link with polypeptides and has good stability, and HYNIC can also form very stable chelates with metal ions and EDDA or Tricine, which can be used as MRI probes.

Embodiment 1, HYNIC and Tricine and the MRI probe of paramagnetic metal Gd

1. Synthesis of GdCl ₃ : Take 36.3 mg of Gd ₂ O ₃ , add it to 10 ml of 0.1 mol/L HCl, and heat to 60°C. Continue to react for 10 minutes until it is completely dissolved, then heat to 100°C to evaporate excess HCl.

2. Preparation of chelating agent solution: dissolve 3mg [HYNIC-D-Phe ¹ , Tyr ³ ]-Octreotide (HYNIC-TOC, synthesized by Shanghai Gil Biochemical Co., Ltd.) in 8ml 0.2M Na ₂ HPO ₄ PH6-7 buffer solution, fully After dissolving, take 20mg Tricine and 50mg mannitol and dissolve them in the above solution, and heat in a water bath for 10 minutes.

3. Preparation of MRI probe: Take the GdCl ₃ (3-4 μmol) synthesized in the first step and add it to the above chelating agent solution for chelation reaction. Gently shake and mix at room temperature. The product is separated by HPLC to obtain Gd-Tricine-HYNIC- Octreotide molecular probe.

The molecular weight of the molecular probe is below 2000.

Slowly inject the synthesized MRI probe—Gd-Tricine-HYNIC-Octreotide (this probe has a high signal on T1WI phase) molecular probe solution through the ear vein into a healthy New Zealand white rabbit that has been completely anesthetized, and no abnormalities are observed. Afterwards, start the MR scan, repeat the scan with the same sequence every half hour, and observe the signal changes of the liver, spleen, kidney, bladder and other organs. The results are shown in Figure 1A-Figure 1F, where Figure 1A is the T1WI images of the rabbit liver before administration, Figure 1B is the T1WI image of the rabbit liver after administration for 0min, Figure 1C is the T1WI image of the rabbit liver after administration for 30min, Figure 1D is the T1WI image of the rabbit liver after administration for 60min, and Figure 1E is the T1WI image of the rabbit liver after administration for 90min, Figure 1F is a T1WI image of rabbit bladder after administration for 90 minutes; Figure 1G is a T1WI image of rabbit kidney scanned immediately after administration. At the same time, the conventional contrast agent Gd-DTPA (Magnevist, Schering, Germany) was used as a control, and the results are shown in Figure 2. Figure 2A is a T1WI image of the rabbit liver before administration, and Figure 2B is a T1WI image of the rabbit liver after administration of 0min. 2C is the T1WI image of rabbit liver after drug administration for 30 minutes; Figure 2D is the T1WI image of rabbit liver after drug administration for 60 minutes; Figure 2E is the T1WI image of rabbit bladder after drug administration for 60 minutes; Immediately scan the T1WI images of the rabbit kidneys.

The results show that the MRI probe of the present invention is different from conventional non-specific contrast agents and has the following characteristics:

1. After the drug injection, the T1WI signal of the liver and spleen was higher than before the drug injection, and it was most significant at 60 minutes (Fig. 1D), indicating that the normal liver and spleen had the distribution of the probe of the present invention; With the lapse of injection time, the signal decreased.

2. The signal of the gallbladder was very low before administration and at 0 min, and gradually showed high signal after 30 min, which suggested that the probe was mainly excreted through the liver and gallbladder; the conventional contrast agent (Magnevist) was mainly excreted through the kidney.

3. During the whole inspection process, no obvious vascular enhancement shadow was seen (may be related to the slow injection in this experiment). The specificity of the probe of the present invention is a non-vascular contrast agent, which is different from conventional contrast agents.

4. At 90 minutes, there was no obvious enhancement in the bladder (Fig. 1F). At this time, the conventional contrast agent (Magnevist) had the highest concentration in the bladder and the strongest signal (Fig. 2F).

5. There was no obvious enhancement in T1 of the kidney (Fig. 1G); however, the enhancement of the kidney with conventional contrast agent (Magnevist) was obvious (Fig. 2G).

Based on the above observation results, it can be seen that Gd-Tricine-HYNIC-Octreotide has different metabolic and in vivo characteristics from conventional contrast agents (Magnevist), and is a specific contrast agent.

Embodiment 2, HYNIC and EDDA and the MRI probe of paramagnetic metal Gd

1. Synthesis of GdCl ₃ : Take 36.3 mg of Gd ₂ O ₃ , add it to 10 ml of 0.1 mol/L HCl, and heat to 60°C. Continue to react for 10 minutes until it is completely dissolved, then heat to 100°C to evaporate excess HCl.

2. Prepare chelating agent solution: dissolve 3mg [HYNIC-D-Phe ¹ , Tyr ³ ]-Octreotide (HYNIC-TOC) in 8ml 0.2M Na ₂ HPO ₄ PH6—7 buffer solution, take 40mg EDDA, 50mg of mannitol was dissolved in the above solution and heated in a water bath for 10 minutes.

3. Preparation of MRI probe: Take the GdCl ₃ (3-4 μmol) synthesized in the first step, add it to the above-mentioned chelating agent solution for chelating reaction, shake gently at room temperature and mix well, and separate the product by HPLC to obtain Gd-EDDA-HYNIC- Octreotide molecular probe.

Through animal experiments, it has the same performance as the Gd-Tricine-HYNIC-Octreotide molecular probe in Example 1.

Claims (7)

1.MRI molecular image probe, comprise central ion, specific molecular and chelating agen, described specific molecular is connected with chelating agen, central ion and chelating agen form chelate, it is characterized in that: described chelating agen comprises main sequestering agent and assistant sequestering agent, described main sequestering agent is a diazanyl crosslinking amino nicotiamide, and described assistant sequestering agent is selected from a kind of in N-three (methylol) methylglycine and the EDDA.

2. MRI molecular image probe according to claim 1 is characterized in that: the mol ratio of diazanyl crosslinking amino nicotiamide and N-three (methylol) methylglycine is 1:2; Perhaps, the mol ratio of diazanyl crosslinking amino nicotiamide and EDDA is 1:1.

3. MRI molecular image probe according to claim 1 and 2 is characterized in that: described central ion is Gd ³⁺, Mn ²⁺, Fe ²⁺Or Fe ³⁺

4. MRI molecular image probe according to claim 1 and 2 is characterized in that: described specific molecular is octreotide or RGD peptide.

5. the preparation method of the described MRI molecular image probe of claim 1 comprises the steps:

1) preparing centre solion;

2) at the Na that is dissolved in pH6-7 ₂HPO ₄Buffer is connected with in the diazanyl crosslinking amino nicotinamide soln of specific molecular, adds N-three (methylol) methylglycine or EDDA and mannitol, mixes, and obtains chelating agent solution;

3) chelating agent solution is mixed with central ion solution, obtain described MRI molecular image probe.

6. preparation method according to claim 5 is characterized in that: central ion is Gd ³⁺, Mn ²⁺, Fe ²⁺Or Fe ³⁺

7. preparation method according to claim 5 is characterized in that: described specific molecular is octreotide or RGD peptide.

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