Nuclear magnetic resonance imaging contrast agent GH-Gd as well as preparation method and application thereof

Abstract

The invention discloses a nuclear magnetic resonance imaging contrast agent GH-Gd as well as a preparation method and application thereof, and belongs to the multidisciplinary crossing fields of material science, kidney pathology, medical imaging and the like. The structure of the contrast agent GH-Gd is shown in the following formula, and the preparation method of the contrast agent GH-Gd comprises the following steps: adding a compound H-DOTA, GGT-1 and cesium carbonate into anhydrous acetonitrile, and carrying out heating reflux reaction to obtain a compound GH-DOTA; and carrying out chelation reaction on the compound GH-DOTA and gadolinium chloride to obtain the nuclear magnetic resonance imaging contrast agent GH-Gd. The contrast agent GH-Gd is formed by chelating a ligand GH-DOTA with Gd < 3 + >, the contrast agent sequentially plays three functions of kidney integration glomerular filtration, GGT and MPO programmed activated kidney enrichment and enzymatic hydrolysis kidney clearance, and the content and dynamic distribution information of GGT and MPO can be obtained while kidney injury is avoided. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the interdisciplinary fields of materials science, nephrology, medical imaging, etc., and particularly relates to a magnetic resonance imaging contrast agent GH-Gd and its preparation method and application. Background Art

[0002] Research has found that γ-glutamyl transpeptidase (GGT) and myeloperoxidase (MPO) are key markers for early acute kidney injury (AKI) and play important roles in the occurrence and development of AKI. Optical detection of γ-glutamyl transpeptidase (GGT) or myeloperoxidase (MPO) has been widely used for the early non-invasive diagnosis of AKI, but the penetration ability of light in tissues is limited, making it difficult to be clinically applied. Secondly, the onset speed of AKI is fast and the mechanism is complex. It is difficult for a single biomolecule to accurately reflect the overall kidney involvement. Sequentially detecting markers of two different pathological states can more comprehensively diagnose the occurrence and development of AKI and improve the diagnostic accuracy. A series of MRI contrast agents that separately detect GGT or MPO have been reported for the diagnosis of cancer, fibrosis, chronic enteritis, etc., but there is no MRI for sequentially detecting renal GGT and MPO to accurately reflect the occurrence and progression of AKI.

[0003] Therefore, it is necessary to develop a magnetic resonance imaging contrast agent GH-Gd that can solve the above technical problems. Summary of the Invention

[0004] The object of the present invention is to provide a magnetic resonance imaging contrast agent GH-Gd and its preparation method and application. The magnetic resonance imaging contrast agent GH-Gd integrates three functions: glomerular filtration, kidney enrichment with programmed activation of GGT and MPO, and enzymatic renal clearance. While avoiding kidney damage, it can obtain the content and dynamic distribution information of GGT and MPO. Based on this, an early non-invasive imaging diagnostic method for acute kidney injury is developed, providing new ideas for the prevention and treatment plans of high-risk patients, and having important clinical significance for the treatment and recovery of kidney injury.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a magnetic resonance imaging contrast agent GH-Gd is provided, and the structural formula of the contrast agent is as follows:

[0007]

[0008] In the second aspect of the present invention, a preparation method of the above magnetic resonance imaging contrast agent GH-Gd is provided. The reaction formula of the preparation method is as follows, and specifically includes:

[0009] (1) Add compound H-DOTA, compound GGT-1, and a base catalyst to an organic solvent, and carry out a heating reflux reaction to obtain compound GH-DOTA;

[0010] Among them, the structural formulas of compound H-DOTA and compound GGT-1 are as follows:

[0011]

[0012] (2) Carry out a chelation reaction between compound GH-DOTA and gadolinium chloride to obtain a magnetic resonance imaging contrast agent GH-Gd.

[0013] In step (1), the organic solvent includes one or more of acetonitrile, dimethylformamide, tetrahydrofuran, dioxane, etc.

[0014] In step (1), the base catalyst includes one or more of cesium carbonate, potassium carbonate, sodium carbonate, rubidium carbonate, etc.

[0015] In step (1), the heating reflux reaction is preferably carried out under a nitrogen atmosphere and in the dark, the reaction temperature is preferably 55 - 85 °C, and the reaction time is preferably 8 - 24 h.

[0016] In step (1), after the reaction, a purification operation is also included. The purification is preferably filtration after the heating reflux reaction, column chromatography purification, and then stirring with a mixed solution of trifluoroacetic acid and dichloromethane, followed by column chromatography purification again.

[0017] In step (1), the molar ratio of compound H-DOTA, compound GGT-1, and the base catalyst is preferably 1:1 - 2:1 - 3.

[0018] In step (2), the chelation reaction between compound GH-DOTA and gadolinium chloride is preferably carried out in a buffer solution. The buffer solution includes sodium acetate buffer solution, phosphate buffer solution, etc.

[0019] In step (2), the chelation reaction is preferably carried out under a nitrogen atmosphere, the reaction temperature is preferably 18 - 35 °C, and the reaction time is preferably 8 - 24 h.

[0020] In step (2), the molar ratio of compound GH-DOTA to gadolinium chloride is preferably 1:1 - 3.

[0021] In the third aspect of the present invention, there is provided the use of the above-mentioned nuclear magnetic resonance imaging contrast agent GH-Gd, and the use includes at least one of the following uses: the use in preparing an imaging tool for detecting acute kidney injury, the use in preparing an MRI contrast agent for simultaneously detecting GGT and MPO, the use in preparing an imaging tool for detecting cancer, the use in preparing an imaging tool for detecting liver fibrosis, and the use in preparing an imaging tool for detecting chronic enteritis.

[0022] The present invention has the following technical effects or advantages:

[0023] The nuclear magnetic resonance imaging contrast agent GH-Gd of the present invention is composed of four different components: 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) group, gadolinium ion (Gd 3+ ), 5-hydroxytryptamine (5-HT) and a GGT-cleavable γ-glutamic acid group (γ-glutamic acid). The group of 5-HT is the substrate of MPO and can specifically bind to the 5-HT receptor highly expressed in renal tubular epithelial cells (PTECs). After 5-HT is covalently bound to DOTA to form H-Gd, the hydroxyl group in H-Gd continues to react with the self-destructive linker. This linker is connected to the GGT-cleavable γ-glutamic acid group to block the activity of the hydroxyl group in H-Gd. In the presence of GGT, the amide bond of γ-glutamic acid is broken, exposing the hydroxyl group of 5-HT to form H-Gd. H-Gd is specifically taken up by PTECs by binding to the 5-HT receptor, increasing the Gd 3+ concentration in the kidney, thereby resulting in the enhancement of T1-weighted magnetic resonance imaging (T1WI) in the kidney region. With the progression of AKI, the gradually increasing MPO and H2O2 in the kidney oxidize the hydroxyl group in H-Gd to form oligomers or generate adducts with nearby proteins or lipid membranes, by increasing the retention of Gd 3+ in the kidney and reducing Gd 3+The tumbling effect further enhances renal T1WI. Finally, under the action of intracellular esterase, DOTA-Gd slowly dissociates from the protein-probe conjugate and is excreted from the kidney, preventing long-term probe enrichment from damaging the kidney and achieving renal clearance of the probe. The present invention utilizes the property that the 5-HT group, the substrate of MPO, can specifically bind to the highly expressed 5-HT receptor in PTECs, and the property that the inactive ester bond can be slowly hydrolyzed under the action of esterase, and innovatively designs a programmed self-fixing and renal clearance magnetic resonance imaging contrast agent GH-Gd, integrating three functions of glomerular filtration, programmed activation of GGT and MPO for renal enrichment, and enzymatic renal clearance, which act in sequence to non-invasively obtain the content and dynamic distribution information of GGT and MPO in the kidney in vivo, while avoiding kidney damage. Based on this, a specific quantitative imaging method for renal GGT and MPO can be developed, making up for the shortcoming of current MRI for sequential imaging of renal GGT and MPO. It provides a new strategy for early non-invasive imaging diagnosis of acute kidney injury and also provides an imaging tool for systematically studying the mechanism of action of dynamic changes in GGT and MPO in acute kidney injury. Description of the Drawings

[0024] Figure 1 is the synthesis route of the contrast agent GH-Gd.

[0025] Figure 2 is the 1H NMR spectrum of the prepared GH-DOTA.

[0026] Figure 3 is the ultraviolet absorption of the solutions of GH-Gd, H-Gd, and the commercial contrast agent Magnevist at 350 nm after being treated with GGT, MPO, and GGT + MPO respectively.

[0027] Figure 4 is the particle size detected by dynamic light scattering (DLS) of the solutions of GH-Gd, H-Gd, and the commercial contrast agent Magnevist at 350 nm after being treated with GGT, MPO, and GGT + MPO respectively.

[0028] Figure 5 are the coronal (A) and axial (B) magnetic resonance images of the kidneys of mice at different time points after intravenous injection of GH-Gd after cisplatin administration, and the relative signal-to-noise ratios of the renal parenchyma (C) and renal pelvis (D) obtained from Figure (A).

[0029] Figure 6 are the coronal (A) and axial (B) magnetic resonance images of the kidneys of mice at different time points after intravenous injection of the commercial Magnevist after cisplatin administration, and the relative signal-to-noise ratios of the renal parenchyma (C) and renal pelvis (D) obtained from Figure (A). Detailed Description of the Invention

[0030] In the following, the present invention will be specifically described in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention.

[0031] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any conflict, this specification shall prevail.

[0032] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0033] As an alternative embodiment of the present invention, the present invention provides a method for preparing a magnetic resonance imaging contrast agent GH-Gd, comprising the following steps:

[0034] (1) DOTA-NHS, 5-hydroxytryptamine hydrochloride, DIPEA, and anhydrous DMF are respectively added into a dry three-necked flask. After reacting for a period of time, the product is collected in a glass bottle to obtain compound H-DOTA.

[0035]

[0036] (2) Compound H-DOTA and compound GGT-1 are added into anhydrous acetonitrile. Finally, cesium carbonate is added. After heating under reflux for a period of time, filtration and purification are carried out; trifluoroacetic acid and dichloromethane are added to the purified product, and after stirring and reacting for a period of time, purification is carried out again to obtain compound GH-DOTA.

[0037]

[0038] Among them, the compound GGT-1 can be prepared according to the method described in the literature Fluoro-Photoacoustic Polymeric Renal Reporter for Real-Time Dual Imaging of Acute Kidney Injury (Penghui Cheng, Kanyi Pu; Methods in Enzymology, 2021:657:271-300.), specifically: BOC-GLU-OTBU (0.7 mmol, 0.212 g) is dissolved in 5 mL of anhydrous dichloromethane. 5 mL of anhydrous dichloromethane dissolving EEDQ (2.8 mmol, 0.691 g) and p-aminobenzyl alcohol (2.8 mmol, 0.344 g) is added to the above reaction solution. The reaction is protected by nitrogen for 12 h, then PBr3 (0.7 mmol, 0.189 g) is added, and the reaction continues for 12 h to obtain the compound GGT-1.

[0039]

[0040] (3) GH-DOTA is dissolved in sodium acetate buffer solution, and then GdCl3·6H2O is added to the above solution and stirred for reaction for a period of time to obtain GH-Gd.

[0041]

[0042] As one of the preferred embodiments of the specific implementation, in step (1), the reaction is carried out in a nitrogen atmosphere for 24 h.

[0043] As one of the preferred embodiments of the specific implementation, in step (1), the dosage ratio of DOTA-NHS, 5-hydroxytryptamine hydrochloride, DIPEA and anhydrous DMF is 0.5 g: 1.0 g: 2.0 g: 10 mL.

[0044] As one of the preferred embodiments of the specific implementation, in step (2), the reaction is a heating reflux reaction carried out at 85 °C under a nitrogen atmosphere and in the dark, and the reaction time is 24 h; the purification is column chromatography purification.

[0045] As one of the preferred embodiments of the specific implementation, in step (2), the dosage ratio of anhydrous acetonitrile, compound H-DOTA, compound GGT-1, and cesium carbonate is 5 ml: 0.1 mmol (56 mg): 0.2 mmol (94 mg): 0.3 mmol (100 mg).

[0046] As one of the preferred embodiments of the specific implementation, in step (2), the stirring reaction time is 5 h; the purification is column chromatography purification.

[0047] As one of the preferred embodiments of the specific implementation, in step (2), the dosage ratio of trifluoroacetic acid to dichloromethane is 20 mL:20 mL.

[0048] As one of the preferred embodiments of the specific implementation, in step (3), the reaction is carried out under a nitrogen atmosphere, and the stirring time is 16 h.

[0049] In the above method, for the reactions involved, if not otherwise specified, they are all carried out at room temperature (18 - 35 °C).

[0050] The contrast agent GH-Gd provided by the present invention contains an inert ester bond with high sensitivity to GGT and MPO and low sensitivity to esterase.

[0051] Next, an NMR imaging contrast agent GH-Gd of the present application, its preparation method and application will be described in detail with reference to examples, comparative examples and experimental data.

[0052] Example 1: An oxidation-responsive self-immobilizing and renal-clearing magnetic resonance imaging contrast agent and its preparation method

[0053] 1. First, prepare the ligand GH-DOTA of the water-soluble programmed GGT and MPO-responsive self-immobilizing and enzymatically cleaved renal-clearing contrast agent, and its structure was characterized by 1H NMR spectroscopy, as shown in Figure 2 shown. The preparation method of the GH-DOTA is as follows:

[0054] (1) 0.5 g of DOTA-NHS, 1.0 g of serotonin hydrochloride, 2.0 g of DIPEA and 10 mL of anhydrous DMF were respectively added to a dry three-necked flask. The mixed solution was reacted for 24 h under nitrogen protection to obtain the compound H-DOTA, which was collected in a glass bottle and sealed.

[0055] (2) Under nitrogen conditions, 0.1 mmol (56 mg) of the compound H-DOTA, 0.2 mmol (94 mg) of the compound GGT-1 were added to 5 mL of anhydrous acetonitrile, and finally 0.3 mmol (100 mg) of cesium carbonate was added. The mixture was heated under reflux at 85 °C for 24 h in the dark and filtered. After column chromatography purification, 20 mL of trifluoroacetic acid and 20 mL of dichloromethane were added to the product, and the mixture was stirred at room temperature for 5 h and then purified by column chromatography to obtain GH-DOTA.

[0056] 2. Under nitrogen protection, GdCl3 was added to the sodium acetate buffer solution containing the ligand GH-DOTA to chelate Gd with the ligand 3+The contrast agent GH-Gd was formed. Specifically: GdCl3·6H2O (1.5 mmol, 1.5 eqv.) was added to 20 mL of sodium acetate buffer solution (1 M, pH = 8) containing compound GH-DOTA (0.99 mmol, 1.0 eqv.), and the mixture was stirred for 16 h to obtain the contrast agent GH-Gd.

[0057] Meanwhile, H-Gd was used as a comparison. The synthesis of H-Gd is shown in the following reaction formula, and its preparation was as follows: GdCl3·6H2O (1.5 mmol, 1.5 eqv.) was added to 20 mL of sodium acetate buffer solution containing compound H-DOTA (0.99 mmol, 1.0 eqv.), and the mixture was stirred for 16 h to obtain H-Gd.

[0058]

[0059] Experimental Example 1

[0060] The contrast agents GH-Gd (500 μM), H-Gd (500 μM), and Magnevist (500 μM) in Example 1 were co-cultured with GGT, MPO + H2O2, and GGT + MPO + H2O2, respectively. The final concentration of GGT in the co-culture system was 20 μg / mL, the final concentration of MPO was 0.3 U / mL, and the final concentration of H2O2 was 0.1 mmol / mL. Since 5-HT will generate quinone free radicals under the action of MPO + H2O2 and undergo a polymerization reaction to form large aggregates, resulting in changes in the particle size of the solution and the enhancement of the ultraviolet absorption at 350 nm. As Figure 3 , under the combined action of GGT and MPO + H2O2, the particle size of GH-Gd increased from 0.5 nm to 356 nm, and the polydispersity index (PDI) increased from 0.15 to 3.6. However, the particle size of GH-Gd did not change after being treated with only GGT or MPO + H2O2. After H-Gd was treated with MPO or GGT + MPO + H2O2, its particle size and PDI increased. The commercial contrast agent Magnevist did not change after being treated with GGT, MPO + H2O2, and GGT + MPO + H2O2. The above results show that GH-Gd can only polymerize and cause an increase in particle size when GGT and MPO + H2O2 coexist. As Figure 4 It was found that, consistent with the particle size test, the ultraviolet absorption of GH-Gd gradually increased only when GGT and MPO + H2O2 coexisted. Based on the above results, it can be proved that GH-Gd has programmed GGT and MPO response performance and can be used to detect GGT and MPO.

[0061] Experimental Example 2

[0062] In BALB / c mice untreated with cisplatin, at 0.5 h after injection of 0.1 mmol / kg GH-Gd or Magnevist, the signal intensity of the renal parenchyma on T1WI of the kidneys increased. At 3 h after administration of GH-Gd, the signal-to-noise ratio of the renal parenchyma and renal pelvis decreased to the pre-administration level. The above results indicate that GH-Gd can be enriched in the kidneys and rapidly metabolized, and is suitable for renal MRI.

[0063] At 12, 24, 48, and 72 h after cisplatin administration (20 mg / kg) to BALB / c mice, 0.1 mmol / kg GH-Gd or the commercial MRI contrast agent Magnevist was intravenously injected respectively. As Figure 5 shown, within 2 h after treatment of the mice with GH-Gd, the T1WI signals of the renal parenchyma and renal pelvis of the mice were positively correlated with the administration time. At the same time, the T1WI signals of the renal parenchyma and renal pelvis of the mice were positively correlated with the administration time of cisplatin. At 3 h after administration of GH-Gd, the T1WI signals of both the renal parenchyma and renal pelvis decreased, indicating good metabolism of GH-Gd by the kidneys. The signal of the kidney region in the mice treated with Magnevist showed no difference from that of the mice without cisplatin treatment within 48 h after cisplatin treatment ( Figure 6 ). The above results indicate that GH-Gd can be used for specific detection of kidney injury in mice by MRI, and can be rapidly metabolized by the kidneys to avoid secondary kidney injury and long-term enrichment in the kidneys.

[0064] Therefore, it is feasible to construct a magnetic resonance imaging method for renal GGT and MPO using this contrast agent, and it is expected to achieve the central goal of early non-invasive imaging diagnosis of AKI induced by nephrotoxins, diabetes, drugs, ischemia / reperfusion, etc., and provide an imaging tool for the study of the injury mechanism of acute kidney injury.

[0065] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A nuclear magnetic resonance imaging contrast agent GH-Gd, characterized in that, The structural formula of the contrast agent GH-Gd is as follows:

2. The preparation method of the magnetic resonance imaging contrast agent GH-Gd according to claim 1, characterized in that, It includes: (1) Add compound H-DOTA, compound GGT-1, and a base catalyst to an organic solvent, and carry out a heating reflux reaction to obtain compound GH-DOTA; Among them, the structural formulas of compound H-DOTA and compound GGT-1 are as follows: (2) Carry out a chelation reaction between compound GH-DOTA and gadolinium chloride to obtain the magnetic resonance imaging contrast agent GH-Gd.

3. The preparation method according to claim 2, wherein The organic solvent described includes one or more of acetonitrile, dimethylformamide, tetrahydrofuran, and dioxane.

4. The preparation method according to claim 2, characterized in that, The base catalyst described includes one or more of cesium carbonate, potassium carbonate, sodium carbonate, and rubidium carbonate.

5. The preparation method according to claim 2, characterized in that, In step (1), the heating reflux reaction is carried out under a nitrogen atmosphere and in the dark, the reaction temperature is 55 - 85 °C, and the reaction time is 8 - 24 h.

6. The preparation method according to claim 2, characterized in that, In step (1), after the reaction, a purification operation is also included. The purification is to filter after the heating reflux reaction, carry out column chromatography purification, then add a mixed solution of trifluoroacetic acid and dichloromethane for stirring, and carry out column chromatography purification again.

7. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of compound H-DOTA, compound GGT-1, and the base catalyst is 1:1 - 2:1 - 3.

8. The preparation method according to claim 2, characterized in that, In step (2), the chelation reaction between compound GH-DOTA and gadolinium chloride is carried out in a buffer solution. The buffer solution includes sodium acetate buffer solution and phosphate buffer solution.

9. The preparation method according to claim 2, wherein, In step (2), the molar ratio of compound GH-DOTA to gadolinium chloride is 1:1 - 3.

10. Use of the nuclear magnetic resonance imaging contrast agent GH-Gd according to claim 1, characterized in that, The applications described include at least one of the following applications: application in the preparation of an imaging tool for detecting acute kidney injury, application in the preparation of an MRI contrast agent for simultaneously detecting GGT and MPO, application in the preparation of an imaging tool for detecting cancer, application in the preparation of an imaging tool for detecting liver fibrosis, application in the preparation of an imaging tool for detecting chronic enteritis.

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