Magnetic resonance contrast agent and its preparation method and application

Abstract

The present application relates to the technical field of biological medicine and molecular imaging, and particularly relates to a novel magnetic resonance contrast agent and a preparation method and application thereof. The contrast agent takes thymosin alpha 1 as a parent, and is connected with three same substituents R. R is a metal chelate group, and the metal chelate group is formed by coordination of a chelating agent and a metal ion. The chelating agent can be diethylenetriamine pentaacetic acid, etc., and the metal ion can be selected from one of gadolinium, manganese and iron. The preparation method of the contrast agent comprises the following steps: grafting the chelating agent to thymosin alpha 1 after activation to form a chelation precursor; and performing coordination of the chelation precursor and the metal ion. The contrast agent prepared by the present application has a significant T1 enhancement effect, has lymphatic selectivity and can reduce accompanying venous pollution. The detection result shows that the in vivo animal imaging has better lymphatic system display capability, and provides a forward-looking reference for replacing the existing contrast agent.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biomedicine and molecular imaging, specifically to the field of chemical synthesis technology and lymphatic system magnetic resonance imaging, and is a magnetic resonance contrast agent and its preparation method and application. Background Technology

[0002] The lymphatic system, distributed throughout the body, consists of lymph, lymphatic vessels, lymphatic capillaries, and lymph nodes. It plays a crucial role in the body's immune response and fight against infection. Drug absorption via the lymphatic system avoids the first-pass effect in the liver; therefore, targeted therapy of the lymphatic system has attracted significant attention. Magnetic resonance lymphangiography (MRI) is an imaging method that uses magnetic resonance imaging (MRI) to visualize lymphatic vessels and lymph nodes after injecting a contrast agent subcutaneously or intradermally. Currently, the most widely used MRI contrast agent in clinical practice is a small-molecule paramagnetic gadolinium (Gd) chelate, which can shorten the T1 relaxation time of tissues, providing better contrast at T1-weighted intervals. I Sequence-specific enhancement of lymphatic tissue signals. Small molecule contrast agents, with their low molecular weight and small particle size, can rapidly enter capillary lymphatic vessels and drain to the target lymph nodes after subcutaneous or intradermal injection. However, small molecule contrast agents have some drawbacks: 1. They can penetrate capillary endothelium and enter accompanying veins around lymphatic vessels, interfering with image quality; 2. They lack targeting specificity, resulting in poor lymph node visualization; 3. They have a short retention time in lymph nodes, which is not conducive to contrast agent accumulation. Therefore, the research and development of larger molecular weight contrast agents is of great significance for medical examinations. Summary of the Invention

[0003] In order to solve the above-mentioned problems in the background art, the main objective of the present invention is to provide a magnetic resonance imaging agent and its preparation method.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A magnetic resonance imaging agent comprising a compound with the chemical structure shown in Formula I: Formula I The parent compound is thymosin α1, which consists of 28 amino acids, is a large polypeptide, has an isoelectric point of 4.2, and a molecular formula of C1. 129 H 215 N 33 O 55 It has a molecular weight of 3108.37 and its sequence is highly conserved, as shown below: Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn.

[0005] Thymosin α1 is linked to three identical substituents R, where R is a metal chelate group formed by coordination of a chelating agent and a metal ion. The chelating agent is diethylenetriaminepentaacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 5,8-biscarboxymethyl-11-[2-(methylamino)-2-oxoethyl]-3-oxo-2,5,8,11-tetraazatridecyl-13-carboxylic acid, or diethylenetriaminepentaacetic acid. Acetic acid-bis(methoxyethylamide), bis(hydroxytriacyl)propionic acid, gadotinoic acid, 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, rel-10-[(1R,2S)-2,3-dihydroxy-1-(hydroxymethyl)propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, wherein the metal ion is selected from gadolinium (Gd), manganese (Mn), and iron (Fe).

[0006] In the above technical solution, the ε-amino group of the three lysine residues of the thymosin α1 is covalently linked to three identical substituents R via amide bonds.

[0007] Furthermore, in the above technical solution, the contrast agent has the following structure: .

[0008] A second aspect of the present invention provides a method for preparing the contrast agent, the method comprising the following steps: (1) After activation, the chelating agent is grafted onto thymosin α1 to form a chelating precursor; (2) The chelate precursor is coordinated with metal ions to obtain a magnetic resonance contrast agent.

[0009] In the above technical solution, further, step (1) involves mixing the chelating agent and the acid-binding agent in a first organic solvent, heating the reaction in the first stage until the solution is clear, cooling the reaction, adding an activating reagent, adding a condensing agent after the solution is clear, and carrying out the second stage reaction to obtain the activated chelating agent reaction solution; mixing the reaction solution with thymosin α1 solution to carry out a grafting reaction, and purifying and drying the reaction solution to obtain the chelating precursor. Step (2) involves dissolving the chelate precursor in deionized water, adjusting the pH to the required range, adding a metal compound to react, and then purifying and drying to obtain the magnetic resonance imaging agent.

[0010] In the above technical solution, further, in step (1), the acid-binding agent is one or more of triethylamine, N,N-diisopropylethylamine, pyridine, sodium carbonate, potassium carbonate, cesium carbonate, potassium acetate, and sodium acetate; the molar number of the acid-binding agent is 3-6 times the molar number of the chelating agent; In step (1), the first organic solvent is one of acetonitrile, benzene, toluene, carbon trichloride, chloroform, dimethyl sulfoxide, acetone, N,N-dimethylformamide, and N,N-dimethylacetamide, and the mass of the first organic solvent is 10-30 times that of the chelating agent. In step (1), the activating agent is N-hydroxysuccinimide, and the number of moles of the activating agent is 2-6 times the number of moles of the chelating agent. In step (1), the condensing agent is one or more of thionyl chloride, oxaloyl chloride, phosphorus oxychloride, phosphorus pentachloride, acetic anhydride, 1-propyl cyclophosphine, ethyl methylphosphonic anhydride, N,N'-carbonyldiimidazole, dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; the molar number of the condensing agent is 1.2-6.6 times the molar number of the chelating agent.

[0011] In step (1), the thymosin α1 solution is obtained by mixing thymosin α1 with deionized water and a second organic solvent; the second organic solvent is one of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methanol, ethanol, acetonitrile, and tetrahydrofuran; the mass of the deionized water is 20-100 times that of the polypeptide, and the mass of the second organic solvent is 5-100 times that of the polypeptide; the molar number of thymosin α1 is 0.01-0.09 times the molar number of the chelating agent.

[0012] In step (1), the chelating agent is one or more of the following: diethylenetriaminepentaacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 5,8-biscarboxymethyl-11-[2-(methylamino)-2-oxoethyl]-3-oxo-2,5,8,11-tetraazatridecyl-13-carboxylic acid, diethylenetriaminepentaacetic acid-bis(methoxyethylamide), bishydroxytriacrylic acid, gadotinoic acid, 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, and rel-10-[(1R,2S)-2,3-dihydroxy-1-(hydroxymethyl)propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and its equivalent number is recorded as 1.0.

[0013] In the above technical solution, further, in step (1), the temperature is raised to 40-60 ℃ in the first stage, and after reacting for 25-50 minutes, the temperature is lowered to 5-25 ℃; the reaction temperature in the second stage is 15-35 ℃, and the reaction lasts for 6-12 hours.

[0014] In step (1), the grafting reaction temperature is 15-40 ℃; the reaction time is 8-24 h.

[0015] In the above technical solution, further, in step (2), the metal compound is one of gadolinium nitrate, gadolinium chloride, gadolinium nitrate hexahydrate, gadolinium chloride hexahydrate, ferric chloride, ferric chloride hexahydrate, manganese chloride, and manganese chloride tetrahydrate; the molar number of the metal compound is 3-5 times the molar number of thymosin α1; In step (2), the mass of deionized water used is 20-100 times that of the chelation precursor; the pH value is 5-6, which is adjusted by an acid-base regulator, which is one of glacial acetic acid, 0.1N hydrochloric acid, and citric acid.

[0016] In the above technical solution, the reaction temperature of step (2) is 15-40 ℃ and the reaction time is 16-32 hours.

[0017] Furthermore, all of the above technical solutions are carried out under an inert atmosphere, and the inert gas is either nitrogen or argon.

[0018] The third aspect of the present invention provides the application of the aforementioned contrast agent or the method for preparing the aforementioned contrast agent in the preparation of magnetic resonance imaging formulations.

[0019] Compared with the prior art, the present invention has the following advantages: This invention is the first to use thymosin α1 as the matrix for a contrast agent and specifically link it to a metal chelate. The resulting contrast agent exhibits greater lymphotropy than small-molecule magnetic resonance imaging (MRI) contrast agents, remaining within lymph nodes for a longer period and providing clearer visualization. The contrast agent of this invention has a moderate molecular weight, making it difficult to penetrate vascular endothelial cells and enter blood vessels, thus preventing contamination of accompanying veins and resulting in better image quality. Furthermore, considering economic efficiency, compared to very expensive large-molecule peptides, thymosin α1 has a moderately reduced molecular weight, ensuring lymphoselectivity and a significant T1 enhancement effect while controlling costs, facilitating industrial production and commercialization.

[0020] This invention grafts a metal chelate, such as the preferred DTPA-Gd, onto thymosin α1 to obtain a novel contrast agent with a moderate molecular weight, high stability, and significant relaxation enhancement effect. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the compound prepared in Example 1.

[0022] Figure 2 The image shows the infrared spectrum of the compound prepared in Example 1.

[0023] Figure 3 The image shows the gel permeation chromatography spectrum of the compound prepared in Example 1.

[0024] Figure 4The image shows the relaxation rate of the compound prepared in Example 1.

[0025] Figure 5 This is a magnetic resonance imaging image of subcutaneous adipose tissue.

[0026] Figure 6 This is a magnetic resonance imaging (MRI) image of muscle tissue.

[0027] Figure 7 Magnetic resonance imaging of C57 wild-type mice before subcutaneous injection of contrast agent on the dorsum of the foot; A. Axial T1WI, B. Coronal reconstruction.

[0028] Figure 8 Magnetic resonance imaging (MRI) images of C57 wild-type mice 10 minutes after subcutaneous injection of contrast agent into the dorsum of the foot; A. Axial T1WI, B. Coronal reconstruction. Detailed Implementation

[0029] The specific embodiments of the present invention will be described in detail below. It should be understood that the following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and should be understood to include those approximate. For numerical ranges, the endpoint values ​​of the ranges, the endpoint values ​​of the ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] This invention uses a Thermo Scientific™ Nicolet™ Summit FTIR spectrometer to detect the infrared spectrum of the product.

[0033] This invention uses a Varian 400 M gel permeation chromatograph to detect the number-average molecular weight, weight-average molecular weight, and PDI of the product.

[0034] This invention uses the Agilent ICP-OES 700 to detect the gadolinium content of the product.

[0035] Example 1 The preparation method for magnetic resonance imaging contrast agents is as follows: (1) Synthesis of “active chelating agent” Add 120 mL of acetonitrile, 8.00 g of diethylenetriaminepentaacetic acid (20.34 mmol), and 9.8 g of triethylamine (96.9 mmol) to a 500 mL three-necked flask R1 in a single batch. Start stirring at 300 rpm; the system is currently a turbid solution. Raise the temperature in R1 to 50°C and stir for 25 minutes. As stirring continues, the system gradually dissolves, and the temperature is immediately lowered to 15°C. Add 2.8 g of N-hydroxysuccinimide (24.35 mmol) to the cooled reaction solution in R1 in a single batch; the system is now a suspension. Continue stirring until the solution is completely dissolved. Then add 5.9 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (30.8 mmol) to R1 in a single batch; the system dissolves rapidly. Raise the temperature in R1 to 25°C and react at this temperature for 6 hours to obtain the activated chelating agent solution.

[0036] (2) Synthesis of “chelate precursor” Add 50 mL of deionized water and 20 mL of dimethyl sulfoxide to a 500 mL three-necked flask R2, and then add 2 g of thymosin α1 (molecular weight 3108.37, 0.6435 mmol). Start stirring at 300 rpm; after 5 minutes, the system changes from a turbid liquid to a clear solution, yielding a thymosin α1 solution. Add the chelating agent solution from step (1) dropwise to the thymosin α1 solution in R2. Maintain the temperature in R2 at 25 degrees Celsius for 16 hours until the solution is clear. After the reaction, dialyze the solution using a 1 kDa dialysis membrane at room temperature for 3 days. After dialysis, freeze-dry to obtain 2 g of solid, which is the chelation precursor.

[0037] (3) Synthesis of "Magnetic Resonance Imaging Contrast Agent" Add 60 mL of deionized water to a 100 mL single-necked flask R3, then add 2 g of the chelate precursor obtained in step (2). Start stirring at 300 rpm; after 2 minutes, the system changed from a turbid liquid to a clear solution. Slowly add glacial acetic acid to R3 until the pH = 6.0. Add 1.2 g of gadolinium nitrate hexahydrate (2.66 mmol) to the above solution in R3 all at once. Maintain the temperature in R3 at 25 degrees Celsius for 16 hours, and the system changed from a suspension to a clear solution. After the reaction, dialyze through a 1 kDa dialysis membrane for 2 days, and freeze-dry to obtain 2.4 g of solid, which is the contrast agent.

[0038] The entire preparation process was protected by nitrogen gas.

[0039] Example 2 Structural inspection: The contrast agent prepared in Example 1 was detected by infrared spectroscopy, inductively coupled plasma atomic emission spectrometry (ICP), and gel permeation chromatography (GPC).

[0040] (1) Through infrared spectroscopy Figure 2 It can be proven that the synthesized compound is Figure 1 The structure shown is analyzed as follows: Alkanes: CH4 Antisymmetric stretching: 2963.36 cm -1 CH symmetrical stretching: 2930.98 cm -1 In-plane curvature of CH: 1440.10 cm -1 CC extension: 1119.05 cm -1 ; Primary amine: NH4+ Strength: 3485.65 cm2 -1 In-plane bending of NH: 1093.52 cm -1 NH out-of-plane bending: 846.85 cm -1 859.67 cm -1 Carboxylic acids: OH- Stretching: 2930.98 cm -1 Out-of-plane bending at OH: 929.99 cm -1 CO2 expansion / contraction: 1320.98 cm -1 COO- Symmetrical expansion and contraction: 1404.01 cm -1 COO- Asymmetric stretching: 1547.90 cm -1 ; Amide: NH4+ Strength: 3285.56 cm2 -1 NH bending, CN stretching: 1590.87 cm -1 NH bending and CN expansion / contraction: 1156.64 cm -1 ; Among them, 1547.90cm -1 - 1404.01cm -1 = 143 cm -1 The structure is a typical rare-earth-carboxylic acid bridged coordination region, proving that the structure contains Gd-COO- bidentate chelates and is structurally stable.

[0041] (2) Results of GPC testing Figure 3 Information related to the molecular weight of the synthesized compound can be determined:

[0042] Table 1 and Figure 3The molecular weight and chromatographic results of the synthesized compound show that the desired compound was successfully synthesized.

[0043] (3) The gadolinium content of the synthesized compound can be determined by ICP detection results:

[0044] As shown in Table 2, the gadolinium mass fraction in the contrast agent sample prepared in Example 1 was 13.99% (139868.5 mg / kg), confirming that gadolinium ions ( It was successfully coordinated to the chelate precursor and had a high gadolinium content.

[0045] Performance testing: (1) Relaxation rate r1 detection: The prepared contrast agent samples were used to prepare gradient concentration contrast agent solutions and placed in a 3.0 T magnetic resonance imaging (MRI) system for T1 MRI imaging and T1 mapping. The T1 time was obtained by curve fitting of the T1 mapping. The slope of the curve was obtained by linear fitting of the reciprocal of the T1 relaxation time (1 / T1) and the contrast agent concentration, which is the relaxation rate r1.

[0046] Test results showed that the relaxation efficiency of the prepared contrast agent was r1 = 3.21 mmol. -1 S -1 It is close to the currently used clinical contrast agent Magenvit Gd-DTPA (3.5 mmol). -1 S -1 ),See Figure 4 .

[0047] (2) Magnetic resonance imaging of ex vivo tissue: The synthetic magnetic resonance contrast agent (diluted 10 times) was injected into ex vivo muscle tissue via subcutaneous and intramuscular injection, respectively, at T1W. I See imaging results Figure 5 , Figure 6 This indicates that the contrast agent prepared by this invention has a significant T1 enhancement effect.

[0048] Practical applications: C57 wild-type mice were subcutaneously injected with approximately 20 μL of the contrast agent aqueous solution (diluted 25 times) from Example 1 into the dorsum of the left paw. Axial fat inhibition 3D-T1W was then performed before injection and at 0, 5, and 10 minutes after injection. I The scan below shows comparison images before and 10 minutes after injection: Figure 7 (A) is the transverse axial T1W before injection. I , Figure 7 (B) Coronal reconstruction. Figure 8 (A) is an axial T1W view 10 minutes after contrast agent injection. I , Figure 8 (B) Coronal reconstruction image. Subcutaneous lymphatic vessels on the injection side are visible 10 minutes after injection (arrow location), indicating the lymphatic system-targeted delivery characteristics of the contrast agent of this invention.

[0049] Although the present invention has been described in detail through the preferred embodiments described above, it should be understood that the above description should not be considered as a limitation of the present invention. Those skilled in the art will understand that modifications or adjustments can be made to the present invention based on the teachings of this specification. These modifications or adjustments should also be within the scope defined by the claims of the present invention.

Claims

1. The application of a magnetic resonance contrast agent in the preparation of a formulation for magnetic resonance lymphangiography, characterized in that, The magnetic resonance imaging agent comprises a compound with the chemical structure shown in Formula 1: Formula I The contrast agent is based on thymosin α1, which is linked to three identical substituents R, where R is a metal chelate group. The metal chelate group is formed by coordination between a chelating agent and a metal ion. The chelating agent is diethylenetriaminepentaacetic acid, 5,8-biscarboxymethyl-11-[2-(methylamino)-2-oxoethyl]-3-oxo-2,5,8,11-tetraazatridecyl-13-carboxylic acid, or diethylenetriaminepentaacetic acid-bis(methoxyethylamide). The metal ion is selected from gadolinium. The method for preparing the contrast agent includes the following steps: (1) The chelating agent and the acid-binding agent are mixed in the first organic solvent. The reaction is heated to the point of complete dissolution in the first stage, and then cooled. An activating reagent is added, and after complete dissolution, a condensing agent is added to carry out the second stage reaction to obtain the activated chelating agent reaction solution. The reaction solution is mixed with a thymosin α1 solution to carry out a grafting reaction. After the reaction, the solution is purified and dried to obtain the chelating precursor. The molar amount of thymosin α1 is 0.01-0.09 times the molar amount of the chelating agent. (2) The chelate precursor is dissolved in deionized water, the pH value is adjusted to the required range, a metal compound is added to react, and the mixture is purified and dried to obtain the magnetic resonance contrast agent. In step (1), the temperature is raised to 40-60℃ in the first stage and reacted for 25-50 minutes, then cooled to 5-25℃; the reaction temperature is 15-35℃ in the second stage and the reaction lasts for 6-12 hours. In step (1), the grafting reaction temperature is 15-40℃; the reaction time is 8-24 h. The reaction temperature in step (2) is 15-40℃, and the reaction time is 16-32 hours; the pH value is 5-6.

2. The application according to claim 1, characterized in that, In step (1), the acid-binding agent is one or more of triethylamine, N,N-diisopropylethylamine, pyridine, sodium carbonate, potassium carbonate, cesium carbonate, potassium acetate, and sodium acetate; the molar number of the acid-binding agent is 3-6 times the molar number of the chelating agent. In step (1), the first organic solvent is one of acetonitrile, benzene, toluene, carbon trichloride, chloroform, dimethyl sulfoxide, acetone, N,N-dimethylformamide, and N,N-dimethylacetamide, and the mass of the first organic solvent is 10-30 times that of the chelating agent. In step (1), the activating agent is N-hydroxysuccinimide, and the number of moles of the activating agent is 2-6 times the number of moles of the chelating agent. In step (1), the condensing agent is one or more of the following: sulfoxide, oxaloyl chloride, phosphorus oxychloride, phosphorus pentachloride, acetic anhydride, 1-propyl phosphate cycloanhydride, ethyl methylphosphonic anhydride, N,N'-carbonyldiimidazole, dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; the molar number of the condensing agent is 1.2-6.6 times the molar number of the chelating agent. In step (1), the thymosin α1 solution is obtained by mixing thymosin α1 with deionized water and a second organic solvent; the second organic solvent is one of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methanol, ethanol, acetonitrile, and tetrahydrofuran; the mass of the deionized water is 20-100 times that of the polypeptide, and the mass of the second organic solvent is 5-100 times that of the polypeptide.

3. The application according to claim 1, characterized in that, In step (2), the metal compound is one of gadolinium nitrate, gadolinium chloride, gadolinium nitrate hexahydrate, gadolinium chloride hexahydrate, ferric chloride, ferric chloride hexahydrate, manganese chloride, and manganese chloride tetrahydrate; the molar number of the metal compound is 3-5 times the molar number of thymosin α1; In step (2), the mass of deionized water used is 20-100 times that of the chelation precursor, and it is adjusted with an acid-base regulator, which is one of glacial acetic acid, 0.1N hydrochloric acid, and citric acid.

4. The application according to claim 1, characterized in that, All methods are carried out under an inert atmosphere, with the inert gas being either nitrogen or argon.

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