Development and application of a probe for early diagnosis of thoracic aortic aneurysm and aortic dissection

Abstract

The present invention relates to the development and application of a probe for early diagnosis of thoracic aortic aneurysm and aortic dissection, and specifically provides a compound represented by formula I or a salt thereof, as well as a method for preparing the compound represented by formula I and its application as a probe for early diagnosis of thoracic aortic aneurysm and aortic dissection. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the development and application of an early diagnosis probe for thoracic aortic aneurysm and aortic dissection (TAAD). Background Art

[0002] Thoracic aortic aneurysm and dissection (TAAD) are cardiovascular diseases with an acute onset, a dangerous prognosis, and a high mortality rate. Among them, thoracic aortic aneurysm refers to a vascular disease in which the thoracic aorta is dilated in an irreversible tumor-like manner, resulting in a vascular diameter at the lesion exceeding 50% of the normal value; while aortic dissection refers to acute damage to the aortic intima, blood flow enters the aortic wall from the tear in the intima, and the blood vessel forms a true and false cavity. With the continuous inflow of blood, the false cavity will rapidly extend along the longitudinal axis of the aorta, and in severe cases it may reach the abdominal aorta and iliac arteries. Eventually, the pressure in the false cavity increases sharply, which may lead to aortic wall rupture. TAAD has many and complex causes, unclear pathogenesis, rapid onset, atypical clinical symptoms, and is very easy to be misdiagnosed and mistreated, resulting in a high mortality rate.

[0003] According to research, the mortality rate of patients within 48 hours after aortic tearing is about 40%-50%. In recent years, with the rapid development of medical technology, timely endovascular repair or aortic replacement surgery for TAAD patients can significantly improve the survival rate of patients with dissection. However, due to the insidious onset of TAAD, patients have no obvious symptoms before tearing. Therefore, it is particularly important to find a method for early diagnosis of TAAD before symptoms appear.

[0004] The existing clinical diagnosis of TAAD is mainly based on the degree of aortic dilation or tearing. However, since most TAAD patients develop acute onset and lack significant clinical symptoms before the blood vessels tear, there is currently no early diagnosis method for TAAD. Summary of the invention

[0005] TAAD is a critical vascular disease caused by intimal tears. As the first barrier of blood vessels, endothelial cells play an important role in maintaining vascular function and homeostasis. In our previous studies, we found that in the early stages of TAAD, the barrier function of endothelial cells is destroyed, the distribution of tight junction molecules in the membrane is reduced, and the permeability is increased. Based on this phenomenon of early barrier destruction in TAAD, the inventors synthesized a magnetic resonance contrast agent that can be used for early diagnosis of TAAD.

[0006] To this end, in the first aspect of the present invention, a compound of formula I or a salt thereof is provided,

[0007]

[0008] In a second aspect of the present invention, a method for preparing a compound of formula I or a salt thereof is provided, comprising:

[0009] 1) FITC-labeled dextran (FITC-Dextran) is subjected to a condensation reaction with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to obtain an intermediate FITC-Dextran-DOTA;

[0010] 2) making the intermediate and GdCl 3 6H 2 O undergoes a complexation reaction to obtain a compound shown in formula I.

[0011] In some embodiments, the molecular weight of the FITC-Dextran is 4 kDa.

[0012] In some embodiments, in step 2), the intermediate and the GdCl 3 6H 2 Before the complexation reaction of O occurs, the intermediate is pre-protonated.

[0013] In some embodiments, the protonation is achieved using hydrochloric acid.

[0014] In some embodiments, the concentration of hydrochloric acid is 0.05-0.15 mM, preferably 0.1 mM.

[0015] In some embodiments, in step 1), the condensation reaction is carried out in the presence of a condensing agent and a base.

[0016] In some embodiments, the condensing agent is 1,1-carbonyldiimidazole (CDI).

[0017] In some embodiments, the base is triethylamine (TEA).

[0018] In some embodiments, in step 1), the solvent for the condensation reaction is an anhydrous solvent, preferably anhydrous dimethyl sulfoxide.

[0019] In some embodiments, in step 1), the mass ratio of the FITC-labeled dextran (FITC-Dextran) to the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is 5:2-5:4, preferably 5:3.

[0020] In some embodiments, in step 1), the mass ratio of the FITC-labeled dextran (FITC-Dextran) to the condensing agent is 25:8-5:2, preferably 25:9.

[0021] In some embodiments, in step 1), the mass ratio of the FITC-labeled dextran (FITC-Dextran) to the base is 10:1-10:3, preferably 5:1.

[0022] In some embodiments, in step 1), the ratio of the mass of the FITC-labeled dextran (FITC-Dextran) to the volume of the base (such as triethylamine) is 10 mg:1 μL-5 mg:1 μL, preferably 50 mg:7 μL.

[0023] In some embodiments, in step 1), the condensation reaction is carried out at room temperature, for example, at room temperature for 10-15 h, such as 12 h.

[0024] In some embodiments, after step 1), the method further comprises: purifying the intermediate FITC-Dextran-DOTA. In some embodiments, the purification is performed using a dialysis membrane (eg, MWCO 1000).

[0025] In some embodiments, in step 2), the intermediate and the GdCl 3 6H 2 The mass ratio of O is 10:3-2:1, preferably 5:2.

[0026] In some embodiments, in step 2), the solvent for the complexation reaction is water, preferably deionized water.

[0027] In some embodiments, in step 2), the complexation reaction is carried out at room temperature, for example, at room temperature for 10-15 h, such as 12 h.

[0028] In some embodiments, after step 2), further comprising: purifying the reaction product of the complex reaction; optionally, further comprising: drying (such as freeze drying) the purified product. In some embodiments, the purification is performed using a dialysis membrane (such as MWCO 1000).

[0029] In the third aspect of the present invention, a contrast agent is provided, which comprises the compound represented by the aforementioned formula I or a salt thereof, or a compound or a salt thereof prepared by the aforementioned method.

[0030] In some embodiments, the contrast agent is the compound represented by the aforementioned formula I or a salt thereof, or a compound or a salt thereof prepared by the aforementioned method.

[0031] In some embodiments, the contrast agent is a magnetic resonance contrast agent.

[0032] In some embodiments, the contrast agent is a TAAD targeted magnetic resonance contrast agent.

[0033] In a fourth aspect of the present invention, there is provided the use of the compound represented by the aforementioned formula I or its salt or the compound or its salt prepared by the aforementioned method as a contrast agent or in the preparation of a contrast agent.

[0034] In some embodiments, the contrast agent is a magnetic resonance contrast agent.

[0035] In some embodiments, the contrast agent is a TAAD targeted magnetic resonance contrast agent.

[0036] In the fifth aspect of the present invention, a composition is provided, which comprises the compound represented by the aforementioned formula I or a salt thereof, or a compound or a salt thereof prepared by the aforementioned method, or the aforementioned contrast agent.

[0037] In the sixth aspect of the present invention, a kit is provided, which comprises the compound represented by the aforementioned formula I or its salt, or the compound or its salt prepared by the aforementioned method, or the aforementioned contrast agent.

[0038] In a seventh aspect of the present invention, a method for targeted angiography of a blood vessel with leakage or TAAD is provided, comprising:

[0039] 1) injecting an effective amount of the compound represented by the aforementioned formula I or its salt, or the compound or its salt prepared by the aforementioned method, or the aforementioned contrast agent into a mammal;

[0040] 2) Performing magnetic resonance imaging on the mammal.

[0041] In some embodiments, the methods are used for non-diagnostic or therapeutic purposes (eg, for scientific research).

[0042] In some embodiments, the mammal is a mouse or a human.

[0043] In the eighth aspect of the present invention, provided is the use of the compound of formula I or its salt, or the compound or its salt prepared by the method, or the contrast agent in preparing a reagent for diagnosing (preferably early diagnosis) or predicting TAAD.

[0044] In a ninth aspect of the present invention, a method for diagnosing (preferably early diagnosis) or predicting TAAD is provided, comprising:

[0045] 1) Providing (such as injecting) an effective amount of the compound represented by the aforementioned formula I or its salt, or the compound or its salt prepared by the aforementioned method, or the aforementioned contrast agent to the subject.

[0046] In some embodiments, the method further comprises:

[0047] 2) performing magnetic resonance imaging on the subject.

[0048] In some embodiments, the subject is a mammal, such as a human or a mouse, preferably a human.

[0049] In the tenth aspect of the present invention, provided is the use of the compound of formula I or its salt, or the compound or its salt prepared by the aforementioned method, or the aforementioned contrast agent for diagnosing (preferably early diagnosis) or predicting TAAD.

[0050] In the eleventh aspect of the present invention, provided is the compound of formula I or its salt, or the compound or its salt prepared by the method, or the contrast agent, for use in diagnosing (preferably early diagnosis) or predicting TAAD.

[0051] It should be noted that the aforementioned contrast agent refers to the compound shown in the aforementioned formula I or its salt or the compound or its salt prepared by the aforementioned method. Optionally, it may further contain one or more pharmaceutically acceptable excipients, for example, a solvent, so that the compound shown in the aforementioned formula I or its salt or the compound or its salt prepared by the aforementioned method can be configured into a solution for injection, etc.

[0052] Beneficial effects:

[0053] 1. The present invention can perform non-invasive imaging of leaking blood vessels in mammals (such as mice) by magnetic resonance detection;

[0054] 2. The present invention can predict whether a mammal (such as a mouse) will develop TAAD by magnetic resonance detection. For example, it can predict whether a mouse will develop TAAD in a mouse TAAD model induced by 3-aminopropionitrile fumarate salt (BAPN);

[0055] 3. The magnetic resonance contrast agent of the present invention has no obvious organ toxicity to mammals (such as mice), is highly safe, is simple and quick to synthesize, has low cost and is easily available.

[0056] 4. The present invention can be used as a potential TAAD diagnosis (preferably early diagnosis) probe for mammals (preferably humans or mice). BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1A in the middle shows the aorta of control mice and BAPN-fed mice after Evans blue was injected through the medial canthal vein 5 and 10 days later; B shows the aorta section results of control mice and BAPN-fed mice after dextran was injected through the medial canthal vein 5 and 10 days later.

[0058] Figure 2 A is the synthesis route of the contrast agent; B is the hydrogen spectrum of the synthetic raw material FITC-dextran detected by nuclear magnetic resonance; C is the hydrogen spectrum of the reaction product of FITC-dextran and DOTA detected by nuclear magnetic resonance; D is the chromatographic peak of the synthetic raw material FITC-Dextran in high performance liquid chromatography; E is the chromatographic peak of the reaction product of FITC-Dextran and DOTA in high performance liquid chromatography; F is the gadolinium content in the material after thermogravimetric analysis; G is the linear regression fitting curve of the synthetic contrast agent detected in PBS buffer.

[0059] Figure 3 A in the middle shows HE staining of organ tissue sections of mice and control mice after injection of contrast agent; B shows vascular magnetic resonance imaging and vascular section staining of control mice and BAPN-fed mice before and after injection of contrast agent.

[0060] Figure 4 A in the middle is the vascular magnetic resonance imaging of the control mice and the mice fed with BAPN after injection of contrast agent 5 and 10 days later; B is the brightness enhancement coefficient (improvement) of the vascular wall of the mice fed with BAPN 5 days after injection of magnetic resonance contrast agent relative to the control mice without injection of contrast agent; C is the brightness enhancement coefficient (improvement) of the vascular wall of the mice fed with BAPN 10 days after injection of magnetic resonance contrast agent relative to the control mice without injection of contrast agent; D is the line graph of the brightness enhancement coefficient (improvement) of the vascular wall of the control mice, the mice with TAAD after BAPN feeding and the mice without TAAD after BAPN feeding 5 days and 10 days later; E is the gross picture of the aorta corresponding to the line graph in Figure D; F is the measurement results of the ascending aorta diameter of the control mice and the mice fed with BAPN 5 days and 10 days later; G is the ascending aorta diameter of the mice fed with BAPN 5 days relative to the control mice; H is the ascending aorta diameter of the mice fed with BAPN 10 days relative to the control mice. DETAILED DESCRIPTION

[0061] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0062] The present invention discloses the development and application of a TAAD early diagnosis probe. The present invention discloses a synthesis method of a magnetic resonance contrast agent and its safety and usability evaluation. The present invention further discloses that the magnetic resonance contrast agent can predict the occurrence of TAAD and can be applied to the early diagnosis of TAAD.

[0063] The present invention uses a tight junction tracer molecule as a probe of the contrast agent, connects the complex molecule DOTA, and modifies the molecule with gadolinium to synthesize the contrast agent FMRI-1. In order to explore the early diagnostic effect of the contrast agent on TAAD, a BAPN-induced TAAD model is used, and magnetic resonance detection is performed after contrast agent is injected in the early stage before TAAD is formed in mice, to explore whether mice with contrast effect will develop TAAD later.

[0064] The present invention is further explained below with reference to specific embodiments.

[0065] Example 1: Determination of endothelial barrier damage in the early stage of TAAD

[0066] In the early stage of BAPN-induced TAAD, i.e., 5 and 10 days after feeding mice, the barrier function of the aorta was detected by injecting tracer molecules that diffuse only through the paracellular pathway through the medial angular vein. Figure 1 In A, we can see that in the early stage of TAAD, Evans blue dye leaks out of mice, indicating that endothelial barrier function is impaired in the early stage of TAAD. In addition, by injecting rhodamine B-labeled dextran into the medial canthal vein and observing rhodamine B fluorescence after slicing the blood vessels, the integrity of vascular barrier function can be evaluated. Figure 1 B shows that the vascular barrier is damaged and dextran leaks.

[0067] Example 2: Synthesis and identification of magnetic resonance contrast agents

[0068] 1. Synthesis

[0069] According to Figure 2 FMRI-1 was synthesized in the manner shown in A. Specifically, 100 mg of FITC-labeled dextran (FITC-Dextran) with a molecular weight of 4 kDa was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO), the reaction mixture was stirred at room temperature for 10 min, 60 mg of DOTA, 36 mg of 1,1-carbonyldiimidazole (CDI) and 14 μL of triethylamine (TEA) were added respectively, and complexed at room temperature for 12 h. The crude product was purified using a dialysis membrane (MWCO1000), and the product was FITC-Dextran-DOTA. To prepare the final product FMRI-1, 20 mg of the product of this step was dissolved in 3 mL of deionized water, protonated with 0.1 mM hydrochloric acid, and GdCl was added. 3 6H 2O (8 mg), the reaction mixture was stirred at room temperature for 12 h, and the crude product was purified using a dialysis membrane (MWCO 1000). The final product was freeze-dried to obtain the target compound, which was finally named FMRI-1.

[0070] 2. Identification

[0071] Firstly, the synthetic raw material FITC-dextran and the reaction product of FITC-dextran and DOTA were analyzed by nuclear magnetic resonance hydrogen spectrum, and it was found that the peak between the chemical shift of 5-6ppm in the hydrogen spectrum was broadened ( Figure 2 B, 2C). At the same time, the synthetic raw material FITC-dextran and the reaction product of FITC-dextran and DOTA were detected by high performance liquid chromatography, and it was found that the retention time in the chromatographic peak was reduced (1.98min) due to the increased polarity of the reaction product compared with FITC-dextran ( Figure 2 D, 2E). Both methods above proved that the target compound was successfully synthesized. In addition, the thermogravimetric analysis method was used, that is, in a nitrogen environment, the organic matter was continuously decomposed by heating, and finally the inorganic gadolinium was left behind, so as to determine that each 1 mg of magnetic resonance imaging material contained 21% gadolinium ( Figure 2 F). In addition, contrast agents containing different concentrations of gadolinium ions were prepared in 200 μL of phosphate buffer, and the samples were placed in a nuclear magnetic resonance imaging instrument for scanning. After imaging, the sample 1 / T1 was quantified. Figure 2 G It can be seen that 1 / T1 is linearly related to the concentration of gadolinium ions, and the linear regression coefficient of the synthetic material obtained by calculation, that is, R is 0.9953, indicating that the higher the concentration of the compound, the stronger the imaging ability.

[0072] Example 3: Application of magnetic resonance contrast agents

[0073] 1. Mouse TAAD model establishment and magnetic resonance imaging

[0074] 3-week-old C57BL / 6J male mice were fed a normal diet or a diet containing 0.4% BAPN for 14 days. On days 5 and 10 of modeling, mice were anesthetized with isoflurane and 100 μL of 8 mg / mL FMRI-1 (solvent was saline) was injected into the medial canthal vein. After 5 minutes, vascular T1-weighted imaging was performed using a mouse magnetic resonance scanner. On day 14, the mouse aorta was isolated, and the mortality rate and TAAD incidence of the mice were counted.

[0075] 2. Safety and availability of MRI contrast agents

[0076] 18 days after the mice were injected with the FMRI-1 probe, they were killed and dissected, and the heart, liver, spleen, lung and kidney tissue sections were taken out and HE staining was performed ( Figure 3 A). The results showed that there was no significant difference in the tissues and organs of mice injected with the magnetic resonance probe (i.e., the prepared FMRI-1) and the control mice (i.e., mice fed a normal diet), indicating that FMRI-1 is highly safe. In addition, since the probe not only contains the metal atom gadolinium (Gd) that can be used for nuclear magnetic resonance imaging, it also contains the FITC group that can be imaged under a confocal microscope. In order to explore the usability of the magnetic resonance probe, magnetic resonance detection was performed on the control mice and the mice fed with BAPN for 14 days before and after the injection of the magnetic resonance probe ( Figure 3 B). The results showed that the increase in the vascular wall of the control mice was weak, and there was no obvious FITC fluorescence in the sections. However, after the BAPN-fed mice were injected with FMRI-1, the contrast signal at the vascular wall in the T1 image increased, and the sections also showed that the FITC fluorescence was enhanced compared with the control mice. This shows that FMRI-1 has a targeted contrast effect on TAAD.

[0077] 3. The material can predict the occurrence of TAAD

[0078] Based on the above evaluation of the safety and usability of magnetic resonance contrast agents, the inventors analyzed whether the contrast agent can predict the occurrence of TAAD. 100 μL of 8 mg / mL FMRI-1 (solvent is saline) was injected on the 5th and 10th days of BAPN-induced TAAD, and the mice were sampled 14 days later to preserve the mouse vascular tissue. Figure 4 A shows that after the injection of magnetic resonance contrast agent in the early stage (5th and 10th days) of TAAD in BAPN-induced mice, the contrast signal in the blood vessels of the BAPN group mice was significantly higher than that of the control group mice ( Figure 4 B, 4C). The mice were further divided into three groups according to whether they were fed with BAPN and whether TAAD occurred in the mice after 14 days of BAPN feeding, namely, the control group, the group that did not develop TAAD after 14 days of BAPN feeding, and the group that developed TAAD after 14 days of BAPN feeding. A line graph was drawn for the 5-day angiographic signal increment coefficient and the 10-day angiographic signal increment coefficient for each mouse. ( Figure 4 D, 4E), the increase in the angiographic intensity increment coefficient of the mice that developed TAAD after 14 days of feeding BAPN was significantly increased compared with the control mice group and the group that did not develop TAAD after feeding BAPN (broken line slope). It can be seen that the magnetic resonance contrast agent of the present invention can predict the occurrence of TAAD. In addition, the inventors used magnetic resonance imaging to measure the diameter of the aorta and found that at 5 and 10 days of the BAPN-induced TAAD model, the diameter of the ascending aorta of the mice was not significantly different from that of the control mice ( Figure 4FH), which is consistent with the literature report that most patients with TAAD have no obvious dilation before vascular tearing. This result shows that the magnetic resonance probe of the present invention can predict whether TAAD occurs when there is no obvious dilation of the aorta.

[0079] The above experiment was analyzed using the following statistical methods: (1) Normally distributed continuous random variables were expressed as mean ± standard error (Mean ± SEM); (2) Paired data were analyzed using paired t-test (two-sided test); (3) Unpaired data were analyzed using grouped t-test (two-sided test).

[0080] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that the above embodiments may be modified without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for preparing a compound or a salt thereof, comprising: 1) FITC-labeled dextran (FITC-Dextran) is subjected to a condensation reaction with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to obtain an intermediate FITC-Dextran-DOTA; 2) allowing the intermediate to undergo a complex reaction with GdCl3·6H2O to obtain a compound.

2. The method of claim 1, wherein: In step 2), before the intermediate reacts with the GdCl3·6H2O for complexation, the intermediate is pre-protonated.

3. The method of claim 2, wherein: The protonation is achieved using hydrochloric acid.

4. The method of claim 1, wherein: In step 1), the condensation reaction is carried out in the presence of a condensing agent and a base.

5. The method of claim 4, wherein: The condensation agent is 1,1-carbonyldiimidazole (CDI).

6. The method of claim 4, wherein: The base was triethylamine (TEA).

7. A compound or a salt thereof, prepared by the method according to any one of claims 1 to 6.

8. Use of the compound or salt thereof prepared by the method according to any one of claims 1 to 6 or the compound or salt thereof according to claim 7 in the preparation of a thoracic aortic aneurysm and aortic dissection (TAAD) targeted magnetic resonance contrast agent.

9. A kit comprising the compound or salt thereof prepared by the method according to any one of claims 1 to 6 or the compound or salt thereof according to claim 7.

10. Use of a compound or a salt thereof prepared by the method according to any one of claims 1 to 6 or a compound or a salt thereof according to claim 7 in the preparation of an agent for early diagnosis or prediction of thoracic aortic aneurysm and aortic dissection (TAAD).

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