Application of 1-methyl-L-tryptophan in prevention and / or treatment of thoracic aortic dissection and / or aortic aneurysm

Abstract

The invention relates to application of 1-methyl-L-tryptophan in prevention and / or treatment of thoracic aortic dissection and / or aortic aneurysm. The structural formula of the 1-methyl-L-tryptophan (1-Me-Trp) is as shown in the following formula (1), # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biology, and more specifically, relates to the use of 1-methyl-L-tryptophan in the prevention and / or treatment of thoracic aortic dissection and / or aortic aneurysm. Background Art

[0002] Thoracic aortic aneurysm / dissection is a class of extremely dangerous cardiovascular emergencies with high fatality and disability rates. There is no radical cure method, and the treatment complications remain high. Traditional trans-thoracic or trans-abdominal surgery is the main treatment method. However, after surgical treatment, the perioperative mortality rate is still as high as 18%, and other complications may occur. In addition, for low-risk patients, drug treatment and close observation are often adopted.

[0003] Clinically, the commonly used drugs for the treatment of aortic aneurysm and aortic dissection include adrenaline β-receptor blockers (β-blockers), angiotensin II receptor type 1 antagonists, angiotensin-converting enzyme inhibitors, statins, etc. Among them,

[0004] β-blockers can reduce myocardial contractility and blood pressure, thereby reducing the vascular wall tension, delaying the progression of thoracic aortic aneurysm, and reducing the incidence of vascular rupture and thoracic aortic dissection.

[0005] Angiotensin II receptor type 1 antagonists are a class of commonly used antihypertensive drugs that can inhibit the binding of angiotensin II and its receptor type 1. There are reports indicating that this class of drugs can delay the widening of the aortic root in children with Marfan syndrome, and the application effect is better at a younger age.

[0006] Statins can reduce the occurrence of atherosclerosis and have a protective effect on the arterial wall.

[0007] For the above various drugs, there are also many adverse reactions and side effects clinically. In preclinical studies, the MMP inhibitor doxycycline delays the dilation of the aorta in Fbn1(mgR / mgR) mice by inhibiting MMP2. However, the clinical trial results are still controversial. In short, there is still a difficult situation of no specific treatment drugs for aortic aneurysm and aortic dissection at present. Therefore, there is a need for more specific, safe and effective drugs that can prevent and relieve aortic aneurysm and aortic dissection.

[0008] Based on this, the present invention is proposed. Summary of the Invention

[0009] The present invention first relates to the use of 1-methyl-L-tryptophan (1-Me-Trp) in the preparation of a drug for the prevention and / or treatment of thoracic aortic dissection and / or thoracic aortic aneurysm. The structural formula of the 1-methyl-L-tryptophan (1-Me-Trp) is shown as the following formula (1);

[0010]

[0011] The drugs described above include, but are not limited to, drugs administered through the gastrointestinal tract, drugs administered by intravenous injection, and drugs administered by subcutaneous implantation. Preferably, the drugs are oral administration drugs.

[0012] The beneficial effects of the present invention are as follows.

[0013] 1-Methyl-L-tryptophan (1-Me-Trp) is an inhibitor of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO or INDO). Its D-isomer (D-1MT), also known as Indoximod, is a methylated tryptophan with immune checkpoint inhibitory activity and is currently undergoing clinical trials for cancer treatment, such as advanced melanoma, but there are no reports on its application in other clinical directions.

[0014] In this application, the applicant conducted experiments on the activity of 1-methyl-L-tryptophan (1-Me-Trp) using an animal model and for the first time demonstrated that 1-methyl-L-tryptophan (1-Me-Trp) has the activity of preventing / treating aortic aneurysm and aortic dissection, providing a new potential compound for the drug development of related diseases. Description of the Drawings

[0015] Figure 1 Establishment of an aortic aneurysm / dissection mouse model.

[0016] Figure 2 、 1 Therapeutic effect of 1-methyl-L-tryptophan (1-Me-Trp) on thoracic aortic dissection / aortic aneurysm in a mouse model. Detailed Embodiments

[0017] Experimental Animals, Reagents and Kits, Instruments

[0018] Experimental Animals

[0019] Thoracic aortic dissection / aortic aneurysm model: Male wild-type mice (C57BL / 6) aged 6-8 weeks were purchased from Beijing Weishang Lide Biotechnology Co., Ltd.

[0020] All animals were bred and raised in the SPF-class environmental animal house of the Beijing Institute of Heart, Lung and Blood Vessel Diseases. Wild mice were required to be male mice at 8 weeks of age and weighing about 20-25 g.

[0021] All experimental operations were carried out according to the "Guide for the Care and Use of Laboratory Animals" formulated by NIH in 1996 and the experimental procedures stipulated by the Experimental Animal Management Committee of the Capital Medical University. All experimental animals were grouped by random method.

[0022] The names and suppliers of biochemical reagents and reagent kits are shown in the following table

[0023]

[0024] The names and suppliers of experimental instruments and equipment are as follows

[0025]

[0026]

[0027] Example 1. Construction of a mouse model of aortic dissection / aortic aneurysm (mouse AD model)

[0028] Construction and evaluation of the mouse AD model in the Ang II + BAPN group

[0029] Control group: Male C57BL / 6J mice at 8 weeks of age were given a normal diet

[0030] Aortic dissection model group: Male C57BL / 6J mice at 8 weeks of age were implanted subcutaneously with a micropump for Ang II perfusion (1000 ng / kg / min) for a total of 4 weeks. Among them, 0.15% BAPN feed was given simultaneously in the first 2 weeks, and a normal diet was given in the last 2 weeks

[0031] The evaluation protocol for the effect of inducing aortic dissection / aortic aneurysm in the model was as follows

[0032] 1. On days 7, 14, 21, and 28, ultrasound of the aortic arch and abdomen of live mice was performed to observe whether the aortic wall was regular, measure the aortic diameter, and observe whether the aorta thickened or had a dissection

[0033] 2. After the mice died, HE or elastic fiber staining was performed on the aorta (see Example 2 for the specific method) to observe aortic inflammation; or observe whether there was disorder of the elastic lamina or the presence of aneurysm / dissection

[0034] After 28 days, mouse plasma and aortic arch were collected

[0035] The results showed that

[0036] The process of establishing the mouse aortic dissection / aneurysm model was as shown in Figure 1 A. It could be seen that the tumor formation rate of the mice in the model group reached 93.3%( Figure 1 B), the blood pressure of the mice in the model group increased( Figure 1 C), and compared with the control group, the expression of Ido1 in the aorta of mice with aortic dissection / aneurysm increased( Figure 1 D). The pathological staining of the mouse aorta was as shown in Figure 1 E and Figure 1In group F, no abnormalities were observed in the aorta of the control group mice. In the mice with aortic dissection / aneurysm, the aortic wall was thickened, the number of inflammatory cells increased, and the aortic elastic lamina was severely disrupted.

[0037] Example 2: Therapeutic effect of 1-methyl-L-tryptophan on aortic dissection / aortic aneurysm

[0038] 1-Methyl-L-tryptophan was purchased from Sigma, with the product number 447439. An AD mouse model was constructed using the Ang II infusion + BAPN diet method as described in Example 1. The mice induced with AD were randomly divided into two groups:

[0039] Control group: Given normal drinking water;

[0040] 1-Methyl-L-tryptophan (1-Me-Trp) treatment group: Starting from the first day of establishing the AD model, 1-methyl-L-tryptophan (1-Me-Trp) was added to the drinking water at a concentration of 2 mg / ml for a total of 4 weeks.

[0041] Observation indicators after administration included:

[0042] 1. Body weight: The two groups of mice were weighed weekly. Observe whether the drug affects weight gain.

[0043] 2. 1-Methyl-L-tryptophan (1-Me-Trp) uptake: Observe the drinking water situation of the two groups of mice roughly every day to judge whether the drug affects the change in the water intake of the mice

[0044] 3. Blood pressure change: Measure blood pressure once a week to exclude the effect of the drug on blood pressure and the effect on the formation of mouse aneurysm / dissection due to blood pressure differences.

[0045] Observation endpoint:

[0046] 1. Natural death of mice during the modeling period: Record the death time and cause of death (whether due to rupture of dissection / aneurysm);

[0047] 2. Mice that did not die: All mice were sacrificed on the 28th day of establishing the model, and the blood vessels of all mice were collected for staining, and their blood vessel diameters and dissection incidence rates were counted.

[0048] Preparation of tissue frozen sections:

[0049] At the end of the experiment, the mice were anesthetized and sacrificed with sodium pentobarbital (100 mg / kg). Heparinized normal saline was used for cardiac perfusion to remove the residual blood in the heart. Under a stereomicroscope, the aortic arch and descending aorta were dissected and excised, immersed in 4% paraformaldehyde for 2 hours of fixation, and then transferred to 30% sucrose solution at 4 °C overnight for sufficient dehydration. The next day, the blood vessels were taken out of the sucrose solution, and the tissue moisture was thoroughly blotted with filter paper. The blood vessels were vertically embedded in OCT embedding medium, wrapped with tin foil, and slowly frozen in liquid nitrogen, and could be stored in a -80 °C refrigerator. Serial frozen tissue sections with a thickness of 5 μm were prepared and adhered to positively charged polylysine-coated slides.

[0050] Histopathological examination:

[0051] 1. Observe the elastic lamina by elastic fiber staining:

[0052] The specific operations are as follows:

[0053] (1) After the frozen sections were equilibrated at room temperature, they were fixed with ice-cold acetone and then washed with PBS to remove OCT.

[0054] (2) Incubate with Reagent 3 Elastic Fiber Staining Solution (Elastic Fiber Staining Solution + Weigert's Resorcin-Fuchsin Solution) for 10 minutes and rinse with tap water for 1 minute.

[0055] (3) Incubate with Weigert's Iron Hematoxylin Staining Solution for 5 minutes and rinse with tap water for 1 minute.

[0056] (4) Add 1 drop of Reagent 4 (Picric Acid / Acid Fuchsin Solution) and stain for 2 minutes.

[0057] (5) Wash twice with 70% alcohol (about 30 seconds each time until the sections no longer decolorize), and then rinse slightly with water.

[0058] (6) Dehydrate rapidly with 95% alcohol and then dehydrate with absolute alcohol for 5 minutes.

[0059] (7) Treat with xylene for 5 minutes and mount with neutral resin.

[0060] Photographs were taken using a Nikon Eclipse TE2000-S microscope (Nikon, Tokyo, Japan), and processed using Nikon NIS-ELEMENTS image analysis software. Elastic fibers were black, cell nuclei were dark brown, collagen was red, and muscle was yellow.

[0061] 2. Observe the destruction of the vascular wall structure by HE staining:

[0062] The specific operations are as follows:

[0063] (1) After the frozen sections are equilibrated at room temperature, they are fixed with ice-cold acetone and then the OCT is washed off with PBS;

[0064] (2) Stain with hematoxylin for 2 - 10 minutes and wash off the excess stain with distilled water;

[0065] (3) Differentiate with hydrochloric acid - ethanol differentiation solution for 2 seconds and rinse with running tap water to blue for 5 minutes;

[0066] (4) Stain with eosin staining solution for 30 seconds - 2 minutes and rinse with tap water for 30 seconds to remove the excess stain;

[0067] (5) Dehydration: Immerse in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 2 - 3 seconds each;

[0068] (6) Mounting: Immerse in 100% ethanol for 1 minute; Treat with xylene for 5 minutes and mount with neutral resin;

[0069] (7) Observe under a microscope.

[0070] The results show that:

[0071] In mice with aortic dissection / aneurysm, the mice in the treatment group using 1 - methyl - L - tryptophan (1 - Me - Trp) had a higher survival rate ( Figure 2 A), the formation rate of aortic dissection / aneurysm in the treatment group was reduced ( Figure 2 B), ultrasound showed that the diameters of the aortic arch and abdominal aorta in the treatment group were smaller ( Figure 2 C and Figure 2 D), the gross picture showed less formation of aortic dissection / aneurysm in the treatment group ( Figure 2 E), and the pathological staining of the mouse aorta was as shown in Figure 2 F. Compared with the untreated control group, the thickness of the aortic wall in mice with aortic dissection / aneurysm in the treatment group was reduced, the number of inflammatory cells was decreased, and the fracture of the aortic elastic lamina was alleviated.

[0072] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. Use of 1-methyl-L-tryptophan (1-Me-Trp) in the preparation of a drug for preventing and / or treating thoracic aortic dissection and / or thoracic aortic aneurysm, wherein the structural formula of the 1-methyl-L-tryptophan (1-Me-Trp) is shown in the following formula (1); 2. The use according to claim 1, characterized in that: The drugs include, but are not limited to, drugs administered via the gastrointestinal tract, drugs administered via intravenous injection, and drugs administered via subcutaneous implantation; preferably, the drugs are oral drugs.

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