Mn-based contrast agent as well as preparation method and application thereof

Abstract

The invention relates to a Mn-based contrast agent and a preparation method and application thereof, and the preparation method comprises the following steps: 1) dissolving deferasirox or a derivative thereof in water to obtain a solution a; (2) adding an aqueous solution of manganese salt into the solution a, and sequentially stirring, dialyzing and freeze-drying to obtain a Mn-based complex; 3) dissolving the Mn-based complex in water to obtain a solution b; and 4) adding an aqueous solution of human serum protein into the solution b, and sequentially stirring and performing ultrafiltration to obtain the Mn-based contrast agent, the structural general formula of deferasirox or the derivative thereof is as follows: # imgabs0 #, wherein R is carboxyl or carboxylic acid methyl ester group. Compared with the prior art, the preparation method disclosed by the invention is simple in synthesis process, low in energy consumption and good in environmental compatibility, and the prepared Mn-based contrast agent has relatively high relaxation rate, excellent thermodynamic stability and good biocompatibility and can be used as a T1 magnetic resonance imaging contrast agent for magnetic resonance imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of medical magnetic resonance contrast agents and relates to, in particular to a method for preparing a magnetic resonance Mn-based contrast agent with high thermodynamic stability, high relaxivity and low toxicity. Background Art

[0002] Cancer remains a major disease that endangers people's lives and health and is a major public health problem worldwide. If cancer can be detected and treated as early as possible, the possibility of cure will be greatly improved. Magnetic resonance imaging (MRI) is one of the most commonly used diagnostic methods in clinical practice. With its advantages of non-invasiveness, non-damaging ionizing radiation, and good penetration depth, it plays an increasingly important role in the diagnosis and treatment of tumors. Gadolinium (Gd) has a unique 7 unpaired electrons among lanthanide elements, a long electron relaxation time, and its complexes have good water solubility and thermodynamic stability. Therefore, gadolinium-based complexes are widely used as contrast agents for magnetic resonance imaging. However, recent studies have shown that patients who take gadolinium-based complex contrast agents for a long time are prone to nephrogenic systemic fibrosis. In addition, gadolinium-based complexes slowly dissociate in the body to release Gd 3+ , causing Gd 3+ It accumulates in the human brain and is highly toxic to the body.

[0003] Mn, also a paramagnetic metal ion 2+ Mn is one of the essential elements in biological systems. It contains five unpaired electrons and has a slow electron relaxation rate and a fast water exchange rate. Therefore, magnetic resonance contrast agents represented by Mn-based complexes also show high relaxation rates and imaging effects. 2+ Ions are one of the essential elements of living organisms, but free Mn 2+ Ions have certain toxicity in the body, and excessive Mn 2+ Therefore, the Mn-based complex used as a magnetic resonance contrast agent must not only have a high relaxation rate, but also have sufficiently good thermodynamic stability to prevent the Mn-based complex from dissociating during use and releasing Mn 2+ ions. The problem of insufficient thermodynamic stability of Mn-based complexes can be solved by the precise design of organic ligands in Mn-based complexes. However, the synthesis of such organic ligands requires complicated and tedious synthesis steps, which is not only time-consuming and labor-intensive, but also not suitable for large-scale synthesis. Summary of the invention

[0004] The purpose of the present invention is to provide a Mn-based contrast agent and its preparation method and application, using the chelating agent Deferasirox (DFX) and its derivatives approved by the U.S. Food and Drug Administration (FDA) for the treatment of thalassemia as ligands, through the combination of the chelating agent and Mn2+ The coordination method is used to directly prepare a Mn-based complex with high thermodynamic stability (Mn(DFX-m)n). On this basis, the high affinity between DFX and human serum albumin (HSA) is utilized to form a Mn-based contrast agent (Mn(DFX-m)n@HSA) by encapsulating the Mn(DFX-m)n complex with HSA, which further improves the thermodynamic stability of the complex and improves the water solubility and biocompatibility of the complex.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing a Mn-based contrast agent, the method comprising the following steps:

[0007] 1) dissolving deferasirox or a derivative thereof (DFX-m) in water to obtain a solution a;

[0008] The general structural formula of Deferasirox or its derivatives is as follows:

[0009]

[0010] Wherein, R is a carboxyl group or a methyl carboxylate group;

[0011] 2) adding an aqueous solution of manganese salt to solution a, stirring, dialyzing, and freeze-drying in sequence to obtain a Mn-based complex (Mn(DFX-m)n);

[0012] 3) dissolving the Mn-based complex in water to obtain a solution b;

[0013] 4) Adding an aqueous solution of human serum albumin to solution b, stirring and ultrafiltration were performed in sequence to obtain a Mn-based contrast agent (Mn(DFX-m)n@HSA).

[0014] Furthermore, in step 1), the pH value of the solution a is 5-10. Preferably, the pH value of the solution a is 8.

[0015] Furthermore, in step 2), the manganese salt is an inorganic manganese salt.

[0016] Furthermore, in step 2), the manganese salt is selected from at least one of manganese chloride, manganese nitrate or manganese sulfate. Preferably, the manganese salt is manganese sulfate.

[0017] Further, in step 2), the molar ratio of the manganese salt to deferasirox or its derivative is (0.25-0.5): 1. Preferably, the molar ratio of the manganese salt to deferasirox or its derivative is 0.5:1.

[0018] Further, in step 2), the stirring time is 4-24h and the stirring temperature is room temperature; the dialysis cutoff is 10000Da and the dialysis time is 45-50h; the freeze-drying temperature is -40 to -35°C and the freeze-drying time is 8-24h. Preferably, the stirring time is 12h, the dialysis time is 48h, the freeze-drying temperature is -38°C and the freeze-drying time is 12h.

[0019] Further, in step 3), the pH value of the solution b is 5-10. Preferably, the pH value of the solution b is 8.

[0020] Further, in step 4), the stirring time is 4-24 hours and the stirring temperature is room temperature; in the ultrafiltration, the ultrafiltration cutoff is 10000Da and the ultrafiltration time is 10-20min. Preferably, the stirring time is 12 hours and the ultrafiltration time is 15min.

[0021] Furthermore, in step 4), the molar ratio of the human serum albumin to the Mn-based complex is 1:(25-200). Preferably, the molar ratio of the human serum albumin to the Mn-based complex is 1:25.

[0022] Furthermore, in step 4), the mass ratio of the human serum albumin to the Mn-based complex is (5-10):1.

[0023] A second aspect of the present invention provides a Mn-based contrast agent, which is prepared by the above method.

[0024] A third aspect of the present invention provides a use of a Mn-based contrast agent, wherein the Mn-based contrast agent is used for magnetic resonance imaging.

[0025] Furthermore, the Mn-based contrast agent is T 1 MRI contrast agents are used in magnetic resonance imaging.

[0026] Compared with the prior art, the present invention has the following characteristics:

[0027] 1) The method of the present invention is to prepare Mn(DFX-m) by using the commercially available chelating agent Deferasirox or its derivatives and human serum albumin with metal ions through coordination and assembly. n @HSA, this synthesis method does not require the preparation of small molecule organic ligands through complex synthesis steps, the synthesis process is simple, the energy consumption is low, and the environmental compatibility is good;

[0028] 2) Mn(DFX-m) prepared by the present invention n@HSA contrast agent has a high relaxivity, excellent thermodynamic stability, good biocompatibility, and a simple and efficient preparation method. It can be used in magnetic resonance imaging, medical diagnosis and treatment and other fields. This probe can be used in the field of medical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Mn(DFX-1) prepared in Example 1 n Relaxation rate curve in Tris-HCl solution (pH=7.2) under 1.0T magnetic field;

[0030] Figure 2 Mn(DFX-1) prepared in Example 1 n In Tris-HCl solution (pH = 7.2) under 1.0T magnetic field, T 1 、T 2 Weighted imaging map;

[0031] Figure 3 Mn(DFX-1) prepared in Example 1 n @HSA relaxation rate curve in Tris-HCl buffer solution (pH=7.2) under 1.0T magnetic field;

[0032] Figure 4 Mn(DFX-1) prepared in Example 1 n @HSA in Tris-HCl buffer solution (pH = 7.2) under 1.0T magnetic field T 1 、T 2 Weighted imaging map;

[0033] Figure 5 Chrome black T solution (A), Mn (DFX-1) n (B), Mn(DFX-1) n @HSA(C) and MnCl 2 (D) Photograph in chrome black T solution;

[0034] Figure 6 Mn(DFX-1) prepared in Example 1 n @HSA in mice under 1.0T magnetic field 1 Weighted imaging map. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0036] The following are more detailed implementation cases, which further illustrate the technical solutions of the present invention and the technical effects that can be obtained.

[0037] In the following examples, unless otherwise specified, raw materials, reagents or processing techniques are all conventional commercial products or conventional processing techniques in the art.

[0038] In the following examples, the carboxylic acid methyl ester-substituted deferasirox has a CAS number of 1266741-05-2, Adamas, and a product number of 015129969.

[0039] Embodiment 1:

[0040] A Mn-based contrast agent, the preparation method of which is as follows:

[0041] S1: Deferasirox DFX-1 (186.5 mg, 0.5 mmol) was dissolved in 100 mL of aqueous solution, the pH thereof was adjusted to about 8 by saturated NaOH solution, and 2.5 mL of 0.1 mol / L aqueous solution of manganese chloride was quickly added to the solution to obtain a mixed solution;

[0042] S2: After the mixture was stirred at room temperature for 12 hours, the reaction solution was dialyzed through a dialysis bag with a cutoff of 10,000 Da for 48 hours. The dialyzate was freeze-dried (freeze-drying temperature was -38 °C, freeze-drying time was 12 hours) to obtain a small molecule Mn-based complex, recorded as Mn(DFX-1) n . ;

[0043] S3: 5 mg Mn(DFX-1) n Dissolve in water, adjust the pH to about 8 with saturated NaOH solution, dissolve 25 mg HSA in water and add to Mn(DFX-1) n The solution was stirred at room temperature for 12 hours and ultrafiltered through an ultrafiltration tube (molecular weight cutoff 10000Da) for 15 minutes to obtain a Mn-based contrast agent, which was recorded as Mn(DFX-1). n @HSA.

[0044] For the above Mn(DFX-1) n @HSA performs performance testing:

[0045] The test method is as follows:

[0046] Mn(DFX-1) n The samples were diluted to 0.1, 0.2, 0.3, 0.4, and 0.5 mM using Tris-HCl (pH = 7.2) solution, and r was measured using a 1.0 T magnetic resonance scanner. 1 、r 2 and T1 、T 2 Weighted imaging. Mn(DFX-1) n @HSA was diluted to 0.1, 0.2, 0.3, 0.4, and 0.5 mM using Tris-HCl (pH = 7.2) solution, and r was measured using a 1.0T magnetic resonance scanner. 1 、r 2 and T 1 、T 2 Weighted imaging. Mn(DFX-1) n @HSA was diluted to 1.5 mmol / L using Tris-HCl (pH = 7.2) solution. Balb / c female mice (weight 18-22 g) were anesthetized with 2.5% avertin (100-200 μL / 10 g) and the above contrast agent solution (dose 0.025 mmol / kg) was injected into the tail vein. T values ​​of the kidneys were collected at 0, 15, 30, 60, 120, and 240 minutes after injection. 1 Weighted MRI images.

[0047] The results are as follows:

[0048] Figure 1 Mn(DFX-1) n The relaxation rate curve of Mn(DFX-1) in Tris-HCl solution (pH=7.2) under 1.0T magnetic field. n The longitudinal relaxation rate is r 1 =7.12mM -1 s -1 , transverse relaxation rate r 2 =15.08mM -1 s -1 , proving that it has good relaxation properties.

[0049] Figure 2 Mn(DFX-1) n In Tris-HCl solution (pH = 7.2) under 1.0T magnetic field, T 1 、T 2 Weighted imaging diagram. It can be seen from the figure that the imaging effect is not obvious within the concentration range of 0-0.5mM.

[0050] Figure 3 Mn(DFX-1) n @HSA relaxation rate curve in Tris-HCl buffer solution (pH = 7.2) under 1.0T magnetic field. It can be seen from the figure that Mn(DFX-1) n @HSA longitudinal relaxation rate is r 1 =20.30mM -1 s-1 , transverse relaxation rate r 2 =36.97mM -1 s -1 , proving that HSA can increase the Mn-based complex Mn(DFX-1) n relaxation properties.

[0051] Figure 4 Mn(DFX-1) n @HSA in Tris-HCl buffer solution (pH = 7.2) under 1.0T magnetic field T 1 、T 2 Weighted imaging diagram, from which we can see that at a concentration of 0.2mM, Mn(DFX-1) n @HSA imaging effect is extremely obvious, proving that the imaging effect of the complex is greatly increased after encapsulating HSA.

[0052] Figure 5 Chrome black T solution (A), Mn (DFX-1) n (B), Mn(DFX-1) n @HSA(C) and MnCl 2 (D) Photograph of the chromium black T solution. It can be seen that Mn(DFX-1) n 、Mn(DFX-1) n @HSA preserves the green color of its solution, indicating that the contrast agent is not destroyed by Eriochrome Black T and has high stability.

[0053] Figure 6 Mn(DFX-1) n T1-weighted imaging of @HSA in mice under a 1.0T magnetic field. It can be seen from the figure that the signal intensity in the mouse kidney area is significantly enhanced within 30 minutes of injection, indicating that Mn(DFX-1)n@HSA has excellent renal imaging effect in mice.

[0054] Embodiment 2:

[0055] A Mn-based contrast agent, the preparation method of which is as follows:

[0056] S1: Dissolve carboxylic acid methyl ester-substituted deferasirox DFX-2 (193.5 mg, 0.5 mmol) in 120 mL of aqueous solution, adjust the pH to about 9 with saturated NaOH solution, and quickly add 2.5 mL of 0.1 mol / L aqueous solution of manganese sulfate to the solution to obtain a mixed solution;

[0057] Among them, the structural formula of DFX-2 is as follows:

[0058]

[0059] S2: After the mixture was stirred at room temperature for 12 hours, the reaction solution was dialyzed through a dialysis bag with a cutoff of 10,000 Da for 48 hours. The dialyzate was freeze-dried (freeze-drying temperature was -38 °C, freeze-drying time was 12 hours) to obtain a small molecule Mn-based complex, recorded as Mn(DFX-2) n ;

[0060] S3: 10mg Mn(DFX-2) n Dissolve in water, adjust the pH to about 9 with saturated NaOH solution, dissolve 100 mg HSA in water and add to Mn(DFX-2) n The solution was stirred at room temperature for 12 hours and ultrafiltered through an ultrafiltration tube (molecular weight cutoff 10000Da) for 15 minutes to obtain a Mn-based contrast agent, recorded as Mn(DFX-2)n@HSA.

[0061] In the above embodiment, the chelating agent DFX and its derivatives used for treating thalassemia are used as ligands and Mn 2+ Coordination reaction, first prepare Mn(DFX-m) with high thermodynamic stability n Complex, and then HAS encapsulates Mn (DFX-m) n Preparation of Mn-based contrast agent Mn(DFX-m) by complex n @HSA. The contrast agent of this embodiment is synthesized by direct reaction of existing chelating agents and human serum albumin with metal ions, without the need to synthesize small molecule organic ligands. The synthesis method is simple, energy consumption is low, and environmental compatibility is good. The prepared Mn(DFX-m) n @HSA contrast agent has a high relaxivity, excellent thermodynamic stability, good biocompatibility and low toxicity in Tris-HCl buffer, and can be used in magnetic resonance imaging, medical diagnosis and treatment and other fields.

[0062] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a Mn-based contrast agent, characterized in that: The method comprises the following steps: 1) dissolving deferasirox or a derivative thereof in water to obtain a solution a; The general structural formula of Deferasirox or its derivatives is as follows: Wherein, R is a carboxyl group or a methyl carboxylate group; 2) adding an aqueous solution of a manganese salt to solution a, stirring, dialyzing, and freeze-drying in sequence to obtain a Mn-based complex; 3) dissolving the Mn-based complex in water to obtain a solution b; 4) Adding an aqueous solution of human serum albumin to solution b, stirring and ultrafiltration were performed in sequence to obtain a Mn-based contrast agent.

2. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 1), the pH value of the solution a is 5-10.

3. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 2), the manganese salt is selected from at least one of manganese chloride, manganese nitrate or manganese sulfate.

4. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 2), the molar ratio of the manganese salt to deferasirox or its derivative is (0.25-0.5):

1.

5. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 2), the stirring time is 4-24 hours and the stirring temperature is room temperature; the dialysis cutoff is 10000Da and the dialysis time is 45-50 hours; the freeze-drying temperature is -40 to -35°C and the freeze-drying time is 8-24 hours.

6. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 3), the pH value of the solution b is 5-10.

7. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 4), the stirring time is 4-24 hours and the stirring temperature is room temperature; in the ultrafiltration, the ultrafiltration retention capacity is 10000Da and the ultrafiltration time is 10-20min.

8. The method for preparing a Mn-based contrast agent according to claim 1, characterized in that: In step 4), the molar ratio of the human serum albumin to the Mn-based complex is 1:(25-200).

9. A Mn-based contrast agent, characterized in that It is prepared by the method according to any one of claims 1 to 8.

10. A use of the Mn-based contrast agent as claimed in claim 9, characterized in that: The Mn-based contrast agent is used for magnetic resonance imaging.

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