Use of a trpm11 high-specificity small molecule agonist, ml-sa5

Abstract

This invention discloses the application of ML-SA5, a highly specific small molecule agonist of TRPML1, for the preparation of drugs against aortic aneurysms or aortic dissections. An aortic aneurysm model was constructed, and an aortic dissection model was established using Fbn1 point mutations. The severity of aortic aneurysms or aortic dissections in control and experimental mice was assessed using small animal ultrasound and aortic morphology staining. Results showed that intraperitoneal injection of ML-SA5 or overexpression of TRPML1 significantly reduced abdominal aortic aneurysm / dissection lesions in mice; it also reduced elastic fiber degradation and smooth muscle cell destruction in abdominal aortic aneurysms, decreased the increase in media thickness and collagen fiber deposition in aortic dissections, further improving the progression and histopathological damage of abdominal aortic aneurysms / dissections. This drug showed good therapeutic effects in animal experiments, with high safety and few toxic side effects, and can be used as a drug to inhibit the progression of aortic aneurysms / dissections.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to the application of ML-SA5, a highly specific small molecule agonist of TRPML1. Background Technology

[0002] Transient acceptor potential mucolipin 1 (TRPML1) is a non-selective cation channel located on the lysosomal membrane, primarily permeable to calcium. 2+ Na + It is an isocation that regulates lysosomal function. A highly specific TRPML1 agonist, ML-SA5, has been developed, and its pharmacological effects have been verified in animal models such as Duchenne muscular dystrophy syndrome, Alzheimer's disease, and myocardial ischemia / reperfusion injury. However, there are few reports on the effects of the TRPML1 agonist ML-SA5 on aortic aneurysm / dissection models.

[0003] Aortic diseases include aortic aneurysms and aortic dissections. An aortic aneurysm is a permanent, localized dilation of the aortic wall exceeding 50% of the normal aortic diameter. It can occur in any part of the aorta, but is most common in the abdominal aorta. Abdominal aortic aneurysms have an insidious onset, often without obvious symptoms, and once ruptured, the mortality rate is as high as 80%. Aortic dissection occurs when the intima and media of the aorta tear, allowing blood to enter the media through the intimal tear, eventually forming a false lumen in the arterial wall. Aortic dissection is a dangerous and rapidly progressing disease; approximately 20% of patients die before reaching the hospital. Currently, the main clinical treatment for aortic aneurysms / dissections is surgical intervention, lacking necessary drug treatments to limit disease progression. Surgical treatment is technically demanding, expensive, and prone to postoperative complications. Furthermore, patients still require regular aortic imaging monitoring and long-term anticoagulation therapy post-surgery, significantly reducing their quality of life. Therefore, developing effective intervention drugs for aortic aneurysms / dissections is of significant clinical importance.

[0004] Current research on the pathogenesis of aortic aneurysms / dissections indicates that the main pathological manifestations are inflammatory cell infiltration, vascular smooth muscle cell death, and degradation of collagen and elastic fibers in the arterial wall, leading to arterial degeneration and dissection or dilation. Among these, vascular smooth muscle cell damage and death play a crucial role in aortic aneurysms / dissections. TRPML1 can regulate vascular smooth muscle cell contraction and calcification by modulating lysosomal function, clearing damaged organelles, and repairing cell membrane damage.

[0005] The specific agonist of TRPML1 is ML-SA5 (CAS No.: 2418670-70-7). There are existing reports on its anti-tumor (CN116500272A) and treatment of ulcerative colitis (CN112807309A) medicinal applications, but there are no reports on its application in cardiovascular diseases and aortic aneurysm / dissection. Summary of the Invention

[0006] To address the aforementioned technical challenges, the TRPML1 agonist ML-SA5 was first applied to aortic disease-related conditions: aortic aneurysm and aortic dissection. ML-SA5, administered via intraperitoneal injection, effectively improved the progression of aortic aneurysm / dissection, thus avoiding surgical treatment.

[0007] To achieve the above objectives, this invention first proposes the application of ML-SA5, a highly specific small molecule agonist of TRPML1, in the preparation of drugs for treating aortic aneurysm or aortic dissection.

[0008] Preferably, the aortic aneurysm is an abdominal aortic aneurysm.

[0009] Preferably, the aortic dissection is Marfan syndrome.

[0010] Preferably, the ML-SA5 is administered via intraperitoneal injection.

[0011] The mechanism of action of the highly specific small molecule agonist ML-SA5 of TRPML1 in the treatment of aortic aneurysm / dissection is as follows:

[0012] The TRPML1 agonist ML-SA5 has a good drug safety profile. Intraperitoneal injection of ML-SA5 or overexpression of TRPML1 in mice can significantly reduce the expansion of abdominal aortic aneurysms and improve their progression. At the same time, it reduces the degradation of elastic fibers and the destruction of smooth muscle cells in abdominal aortic aneurysms, further improving the histopathological damage of abdominal aortic aneurysms.

[0013] ML-SA5 can significantly reduce the dilation of the aortic root, thereby improving the progression of aortic dissection; ML-SA5 can significantly reduce the degradation of elastic fibers, the increase in media thickness, and the deposition of collagen fibers in aortic dissection, thereby improving the histopathological damage of aortic dissection.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The present invention uses the TRPML1 ion channel agonist ML-SA5 to administer drugs, which can significantly reduce the dilation of abdominal aortic aneurysms, improve the progression of abdominal aortic aneurysms, and at the same time reduce the degradation of elastic fibers and the destruction of smooth muscle cells in abdominal aortic aneurysms, thereby further improving the histopathological damage of abdominal aortic aneurysms.

[0016] 2. The present invention uses the TRPML1 ion channel agonist ML-SA5 to administer drugs, which can significantly reduce the dilation of the aortic root and thus improve the progression of aortic dissection; ML-SA5 can significantly reduce the degradation of elastic fibers, the increase of media thickness and the deposition of collagen fibers in aortic dissection, thereby improving the histopathological damage of aortic dissection.

[0017] 3. The TRPML1 ion channel agonist ML-SA5 has good drug safety and few toxic side effects, and can be used as a drug to prevent the progression of aortic aneurysm / dissection. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Example 1 is a pair of wild-type mice (Mcoln1) + / + ) and smooth muscle cell-specific overexpression TRPML mice (Mcoln1 smOE An abdominal aortic aneurysm model was constructed by perfusing the abdominal aortic segment with porcine pancreatic protease. Figure 1 A is a simplified diagram of the experimental process. Figure 1 B is an in vitro image of the representative mouse abdominal aorta at the experimental endpoint. Figure 1 C measured the outer diameter of the mouse abdominal aorta using microscopy before perfusion, after perfusion, and at the experimental endpoint. Figure 1 D represents the trend of changes in the outer diameter of the mouse abdominal aorta during the experimental period. Figure 1 E represents the experimental endpoint, a representative mouse abdominal aortic echocardiogram image, with the arrow indicating the abdominal aorta. Figure 1 F represents the internal diameter of the mouse abdominal aorta during the experimental period; Figure 1 G represents the trend of changes in the inner diameter of the abdominal aorta in mice during the experimental period; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0020] Figure 2 Example 1 describes the effects of Apoe knockout on wild-type mice (Apoe) in experiments. - / - ;Mcoln1 + / + ) and smooth muscle cell-specific overexpression TRPML mice (Apoe - / - ;Mcoln1 smOE Construct an abdominal aortic aneurysm model. Figure 2 A is a simplified diagram of the experimental procedure. Figure 2 B is an in vitro image of the representative mouse abdominal aorta at the experimental endpoint. Figure 2 C represents the experimental endpoint, achieved by microscopic measurement of the outer diameter of the mouse abdominal aorta. Figure 2 D represents the trend of changes in the outer diameter of the mouse abdominal aorta during the experimental period. Figure 2 E represents the experimental endpoint, a representative mouse abdominal aortic echocardiogram image, with the arrow indicating the abdominal aorta. Figure 2 F represents the diameter of the mouse abdominal aorta during the experimental period. Figure 2 G represents the trend of changes in the inner diameter of the abdominal aorta in mice during the experimental period. Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0021] Figure 3 The histopathological changes of mice with an abdominal aortic aneurysm model constructed by perfusion of the abdominal aortic segment with porcine pancreatic protease, as described in Experiment 1, are as follows: Figure 3 A shows the HE, EVG, and SMA staining images of the representative mouse abdominal aorta at the experimental endpoint. Figure 3 B represents the experimental endpoint, the score of elastic fiber rupture in the mouse abdominal aorta. Figure 3 C represents the experimental endpoint score for the destruction of smooth muscle cells in the mouse abdominal aorta; compared with the control group, ** indicates P < 0.01;

[0022] Figure 4 The histopathological changes of mice with an abdominal aortic aneurysm model constructed under the Apoe knockout background in Experiment Example 1 are shown below. Figure 4 A shows HE, EVG, and SMA staining images of the representative mouse abdominal aorta at the experimental endpoint. Figure 4 B represents the experimental endpoint, the score for elastic fiber rupture in the mouse abdominal aorta. Figure 4 C represents the experimental endpoint score for the destruction of smooth muscle cells in the mouse abdominal aorta; compared with the control group, ** indicates P < 0.01;

[0023] Figure 5 In Experiment 2, an abdominal aortic aneurysm model was established in C57BL / 6J mice by perfusing the abdominal aortic segment with porcine pancreatic protease and administering ML-SA5. Figure 5 A is a simplified diagram of the experimental procedure. Figure 5 B is an in vitro image of the representative mouse abdominal aorta at the experimental endpoint. Figure 5 C represents the experimental endpoint, achieved by microscopic measurement of the outer diameter of the mouse abdominal aorta. Figure 5 D represents the trend of changes in the outer diameter of the mouse abdominal aorta during the experimental period. Figure 5 E represents the experimental endpoint, a representative mouse abdominal aortic echocardiogram image, with the arrow indicating the abdominal aorta. Figure 5 F represents the diameter of the mouse abdominal aorta during the experimental period. Figure 5G represents the trend of changes in the inner diameter of the abdominal aorta in mice during the experimental period. Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0024] Figure 6 In Experiment 2, an abdominal aortic aneurysm model was constructed under Apoe knockout background and ML-SA5 was administered. Figure 6 A is a simplified diagram of the experimental procedure. Figure 6 B is an in vitro image of the representative mouse abdominal aorta at the experimental endpoint. Figure 6 C represents the experimental endpoint, achieved by microscopic measurement of the outer diameter of the mouse abdominal aorta. Figure 6 D represents the trend of changes in the outer diameter of the mouse abdominal aorta during the experimental period. Figure 6 E represents the experimental endpoint, a representative mouse abdominal aortic echocardiogram image, with the arrow indicating the abdominal aorta. Figure 6 F represents the diameter of the mouse abdominal aorta during the experimental period. Figure 6 G represents the trend of changes in the inner diameter of the abdominal aorta in mice during the experimental period. Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0025] Figure 7 In Experiment 2, an abdominal aortic aneurysm model was established in C57BL / 6J mice by perfusion with porcine pancreatic protease, and histopathological changes were observed in ML-SA5 mice. Figure 7 A shows HE, EVG, and SMA staining images of the representative mouse abdominal aorta at the experimental endpoint. Figure 7 B represents the experimental endpoint, the score for elastic fiber rupture in the mouse abdominal aorta. Figure 7 C represents the score of smooth muscle cell damage in the mouse abdominal aorta as the experimental endpoint; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0026] Figure 8 An abdominal aortic aneurysm model was constructed in Experiment 2 with Apoe knockout, and histopathological changes were observed in ML-SA5 mice. Figure 8 A shows HE, EVG, and SMA staining images of the representative mouse abdominal aorta at the experimental endpoint. Figure 8 B represents the experimental endpoint, the score for elastic fiber rupture in the mouse abdominal aorta. Figure 8 C represents the score of smooth muscle cell damage in the mouse abdominal aorta as the experimental endpoint; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0027] Figure 9In Experiment 3, after modeling by perfusion of the abdominal aorta with porcine pancreatic protease, the serum AST, ALT, white blood cell count, hemoglobin, mean erythrocyte volume and platelet levels of mice in the solvent group and ML-SA5 group were compared.

[0028] Figure 10 This is an example of the lesions in the aortic root and ascending aorta of the Marfan syndrome mouse model (Example 4). Figure 10 A represents the experimental dosing regimen; Figure 10 B represents the experimental endpoint, a representative mouse aortic echocardiogram. Figure 10 C represents the change in the inner diameter of the aortic root in mice aged 3-5 months during the drug administration period; Figure 10 D represents the change in the ascending aorta diameter of mice aged 3-5 months during the drug administration period; Figure 10 E represents the trend of changes in the inner diameter of the aortic root in mice during the experimental period (1-5 months of age); Figure 10 F represents the trend of ascending aortic diameter changes in mice (1-5 months old) during the experimental period; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0029] Figure 11 The image shows the pathological changes in the aortic root of a mouse model of Marfan syndrome (Example 4). Figure 11 A shows the representative mouse aortic EVG and Masson trichrome staining image at the experimental endpoint; Figure 11 B represents the experimental endpoint, the score of the mouse aortic wall structure. Figure 11 C represents the experimental endpoint, the thickness of the medial wall of the mouse aorta. Figure 11 D represents the experimental endpoint, the fraction of collagen deposition in the aortic wall of mice; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0030] Figure 12 The image shows the lesions in the aortic root and ascending aorta of Marfan syndrome model mice after ML-SA5 administration (Example 5). Figure 12 A is the experimental dosing regimen: 2-3 month old mice were intraperitoneally injected with ML-SA5 (2.5 mg / kg / 2d) or the solvent for 8 weeks. Changes in the aortic diameter were monitored by echocardiography at the start, midpoint, and end of the experiment. Figure 12 B represents the experimental endpoint, a representative mouse aortic echocardiogram. Figure 12 C represents the change in the inner diameter of the aortic root in mice during 8 weeks of drug administration; Figure 12 D represents the change in the ascending aorta diameter of mice within 8 weeks of drug administration;

[0031] Figure 13The image shows the pathological changes in the aortic root of mice with Marfan syndrome after administration of ML-SA5 in Experimental Case 5. Figure 13 A shows the EVG staining and Masson trichrome staining images of the representative mouse aorta at the experimental endpoint; Figure 13 B represents the experimental endpoint, the score of the mouse aortic wall structure. Figure 13 C represents the experimental endpoint, the thickness of the medial wall of the mouse aorta. Figure 13 D represents the experimental endpoint, the fraction of collagen deposition in the aortic wall of mice; compared with the control group, *** indicates P < 0.001, and **** indicates P < 0.0001. Detailed Implementation

[0032] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0033] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0034] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0035] Experimental Example 1

[0036] Constructing a mouse model of abdominal aortic aneurysm

[0037] There are two classic methods for constructing animal models of abdominal aortic aneurysms: 1) infusing the abdominal aortic segment with porcine pancreatic protease to form an abdominal aortic aneurysm, with a model period of 14 days; 2) infusing angiotensin II continuously via a micro-osmotic pump to form an abdominal aortic aneurysm in the context of Apoe knockout, with a model period of 28 days. In this invention, both of the above methods are used to construct abdominal aortic aneurysms. The specific steps are as follows:

[0038] (1) Grouping of animals:

[0039] ① Establishing an abdominal aortic aneurysm model using porcine pancreatic protease perfusion of the abdominal aorta: After anesthetizing and opening the abdomen of mice, the abdominal aorta was exposed. Under specific conditions, the abdominal aortic segment was perfused with porcine pancreatic protease (1 U / mL, 150 mmHg, 5 min) under pressure to induce a subrenal abdominal aortic aneurysm.

[0040] Twenty adult male wild-type mice (denoted as Mclon1) were selected. + / + ) and 20 adult male mice that specifically overexpressed TRPML1 in smooth muscle cells (denoted as Mclon1) smOEMice (6 mice / cage) were housed under standard conditions (21–24°C, 60% humidity, 12h:12h light / dark cycle) with free access to water and food. Six Mice were randomly selected. + / + Mice and 6 Mclon1 smOE Mice were used as the sham-operated group (Sham), while the rest were McLyn1. + / + Mice and Mcoln1 smOE Mice were randomly divided into abdominal aortic aneurysm model groups, and were further divided into wild-type mice (Mcoln1) and wild-type mice (Mcoln1). + / + ) and smooth muscle cell-specific overexpression of TRPML1 (Mcoln1) smOE The abdominal aorta segment was perfused with physiological saline (Sham group) or porcine pancreatic protease (model group). The external diameter of the mouse abdominal aorta was measured before perfusion, after perfusion, and at the experimental endpoint (day 14 after perfusion). The internal diameter of the mouse abdominal aorta was measured by ultrasound before perfusion, on day 3 and day 7 after perfusion, and at the experimental endpoint (day 14 after perfusion).

[0041] ② An abdominal aortic aneurysm model was induced in Apoe knockout mice by continuous administration of angiotensin II via a micro-osmotic pump: After anesthetizing and bluntly dissecting the skin of Apoe knockout mice, a micro-osmotic pump containing angiotensin II was implanted (pumping rate of 1000 ng / kg / min, duration of 28 days) to induce an adrenal abdominal aortic aneurysm.

[0042] Thirty adult male Apoe knockout mice (denoted as Apoe) were selected. - / - ;Mcoln1 + / + ) and 30 adult male Apoe knockout, smooth muscle cell-specific overexpression TRPML1 mice (denoted as Apoe - / - ;Mcoln1 smOE Mice (6 mice / cage) were housed under standard conditions (21–24°C, 60% humidity, 12h:12h light / dark cycle) with free access to water and food. Six Apoe mice were randomly selected. - / - ;Mcoln1 + / + Mice and 6 Apoe - / - ;Mcoln1 smOE Mice were used as the sham-operated group, while the rest were used as the Apoe group. - / - ;Mcoln1 + / + Mice and Apoe - / - ;Mcoln1 smOE Mice were randomly divided into abdominal aortic aneurysm model groups, and Apoe knockout mice (Apoe - / - ;Mcoln1 + / + Apoe knockout and smooth muscle cell-specific overexpression TRPML1 mice (Apoe - / -;Mcoln1 smOE Using a micro-osmotic pump, physiological saline (Sham group) or angiotensin II (model group) was continuously infused. The external diameter of the abdominal aorta of mice was measured at the experimental endpoint (day 28), and the internal diameter of the abdominal aorta of mice was measured by ultrasound before the experiment, on day 14 after the experiment, and at the experimental endpoint (day 28).

[0043] (2) Detection of the inner and outer diameters of the abdominal aorta in model mice

[0044] The inner diameter of the abdominal aorta was detected by small animal ultrasound in mice in the Sham group and model group of Experiment Example 1; the outer diameter of the abdominal aorta was detected by microscope and microscopy software.

[0045] Experimental results are as follows Figure 1 and Figure 2 As shown, in both models, smooth muscle cell-specific overexpression of TRPML1 significantly reduced the expansion of abdominal aortic aneurysms, indicating that smooth muscle cell-specific overexpression of TRPML1 improves the progression of abdominal aortic aneurysms.

[0046] (3) Morphological staining method to assess the severity of abdominal aortic aneurysm in model mice

[0047] To further evaluate the effect of smooth muscle cell-specific overexpression of TRPML1 on abdominal aortic aneurysms, the degree of lesions in the abdominal aorta was assessed at the histopathological level using section staining. After the establishment of the model in the Sham group and model group mice in step (1), abdominal aortic aneurysm specimens were taken, paraffin-embedded, sectioned, and histopathologically evaluated using staining techniques. HE staining was used to observe the basic morphology of blood vessels, EVG staining was used to observe the breakage of vascular elastic fibers, and SMA staining was used to observe the damage to vascular smooth muscle cells.

[0048] Experimental results are as follows Figure 3 and Figure 4 As shown, in both models, smooth muscle cell-specific overexpression of TRPML1 significantly reduced elastic fiber degradation and smooth muscle cell destruction in abdominal aortic aneurysms, indicating that smooth muscle cell-specific overexpression of TRPML1 improves the histopathological damage of abdominal aortic aneurysms.

[0049] Experimental Example 2

[0050] A mouse abdominal aortic aneurysm model was established, and ML-SA5 was administered.

[0051] Given that smooth muscle cell-specific overexpression of TRPML1 can improve abdominal aortic aneurysms, we next synthesized the TRPML1 ion channel-specific agonist ML-SA5 and administered it to the two abdominal aortic aneurysm models mentioned above to investigate its effects on abdominal aortic aneurysms. The specific steps are as follows:

[0052] (1) Grouping of animals:

[0053] ① An abdominal aortic aneurysm model was established by perfusing the abdominal aortic segment with porcine pancreatic protease: Fifty adult male wild-type (C57BL / 6J) mice were selected, and mice (6 mice / cage) were housed under standard conditions (21-24℃, 60% humidity, 12h:12h light / dark cycle) with free access to water and food. Twelve mice were randomly selected as the sham operation group (Sham), and were further divided into: Sham + solvent group and Sham + drug administration group (ML-SA5, 10mg / kg / d). The remaining mice were used as the abdominal aortic aneurysm model group, and were further divided into: model + solvent group and model + drug administration group (ML-SA5: 2mg / kg / d; 5mg / kg / d; 10mg / kg / d). Wild-type mice (C57BL / 6J) were perfused with physiological saline (Sham group) or porcine pancreatic protease (model group) in the abdominal aorta. The external diameter of the mouse abdominal aorta was measured before perfusion, after perfusion, and at the experimental endpoint (14 days after perfusion). The internal diameter of the mouse abdominal aorta was measured by ultrasound before perfusion, on day 7 after perfusion, and at the experimental endpoint (14 days after perfusion).

[0054] ② An abdominal aortic aneurysm model was established by continuously administering angiotensin II via a microosmotic pump in the context of Apoe knockout: 40 adult male Apoe knockout mice were selected. - / - Six Apoe mice per cage were housed under standard conditions (21–24°C, 60% humidity, 12h:12h light / dark cycle) with free access to water and food. Twelve Apoe mice were randomly selected. - / - Mice were used as the sham-operated group and were divided into two groups: a Sham + solvent group and a Sham + drug administration group (ML-SA5, 5 mg / kg / d). The remaining Apoe - / - Mice were used as a model of abdominal aortic aneurysm and were divided into a model + solvent group and a model + drug administration group (ML-SA5, 5mg / kg / d). The external diameter of the abdominal aorta of mice was measured at the experimental endpoint (day 28), and the internal diameter of the abdominal aorta of mice was measured by ultrasound before the experiment, on day 14 after the experiment, and at the experimental endpoint (day 28).

[0055] (2) Detection of the diameter of the abdominal aorta after administration of ML-SA5 to a mouse model of abdominal aortic aneurysm

[0056] The inner diameter of the abdominal aorta was detected by small animal ultrasound in mice in Experiment 2 (Sham group and model group); the outer diameter of the abdominal aorta was detected by microscope and microscopy software.

[0057] Experimental results are as follows Figure 5 and Figure 6The results showed that in both models, administration of the TRPML1 agonist ML-SA5 (5 mg / kg / d, 10 mg / kg / d) significantly reduced the expansion of abdominal aortic aneurysms, indicating that the TRPML1 agonist ML-SA5 improves the progression of abdominal aortic aneurysms.

[0058] (3) Morphological staining method to assess the severity of aneurysms after ML-SA5 administration

[0059] To further evaluate the effect of the TRPML1 ion channel agonist ML-SA5 on abdominal aortic aneurysms, the degree of lesions in the abdominal aorta was assessed at the histopathological level using section staining. After the establishment of the model in the Sham group and model group mice in step (1), abdominal aortic aneurysm specimens were taken, paraffin-embedded, sectioned, and histopathologically evaluated using staining techniques. HE staining was used to observe the basic morphology of blood vessels, EVG staining was used to observe the breakage of elastic fibers in blood vessels, and SMA staining was used to observe the damage to vascular smooth muscle cells.

[0060] Experimental results are as follows Figure 7 and Figure 8 The results showed that in both models, administration of the TRPML1 agonist ML-SA5 significantly reduced elastic fiber degradation and smooth muscle cell destruction in abdominal aortic aneurysms, indicating that the TRPML1 agonist ML-SA5 improves the histopathological damage of abdominal aortic aneurysms.

[0061] Experimental Example 3

[0062] Serological testing to assess the toxicity of the TRPML1 agonist ML-SA5

[0063] To assess the safety of the TRPML1 ion channel agonist ML-SA5, we collected whole blood samples from mice in the porcine pancreatic protease perfusion model + solvent group and the model + ML-SA5 (10 mg / kg / d) group for complete blood count.

[0064] Test results as follows Figure 9 As shown, there was no significant difference in blood routine tests between the ML-SA5 group and the solvent group, indicating that the TRPML1 agonist ML-SA5 has good drug safety.

[0065] Experiment Example 4

[0066] Constructing a mouse model of Marfan syndrome

[0067] The mice used were heterozygous mice with a C-to-G mutation at position 1039 of the Fbn1 gene (denoted as Fbn1). C1039G / + To induce Marfan syndrome. The specific steps are as follows:

[0068] (1) Grouping of animals:

[0069] Seven male wild-type mice (denoted as Fbn1) were selected. + / + ;Mcoln1 + / + ) and 7 male smooth muscle cell-specific TRPML1 overexpressing mice (denoted as Fbn1) + / + ;Mcoln1 smOE Seven male Marfan syndrome mice (denoted as Fbn1) were selected as the sham-operated group. C1039G / + ) and 7 Marfan syndrome mice with male smooth muscle cells specifically overexpressing TRPML1 (denoted as Fbn1) C1039G / + ;Mcoln1 smOE As the model group, mice (6 mice / cage) were housed under standard conditions (21–24°C, 60% humidity, 12h / 12h light / dark cycle) and provided with free access to water and food. All mice in the above groups underwent ultrasound examination of the aortic root and ascending aorta diameter for 2 months starting at 1 month of age, and were then intraperitoneally injected with solvent or ML-SA5 (2 mg / kg / 2d) for 2 months at 3 months of age. The overall experimental period was 4 months.

[0070] (2) Detection of the aortic root and ascending aorta inner diameter in model mice

[0071] Small animal ultrasound was used to detect the aortic root and ascending aortic diameter in mice in the Sham group and model group in Example 4.

[0072] Experimental results are as follows Figure 10 As shown, smooth muscle cell-specific overexpression of TRPML1 can significantly reduce aortic root dilation, indicating that smooth muscle cell-specific overexpression of TRPML1 improves the progression of aortic dissection.

[0073] (3) Morphological staining method to assess pathological changes of aortic dissection in model mice

[0074] To further evaluate the effect of smooth muscle cell-specific overexpression of TRPML1 on aortic dissection, the degree of vascular lesions was assessed at the histopathological level using section staining. After the experiments in the Sham group and model group mice in step (1), specimens from the aortic root and ascending aorta were taken for paraffin embedding and sectioning, and histopathological evaluation was performed using staining techniques. EVG staining was used to observe the breakage of elastic fibers and the thickness of the vascular media, and Masson staining was used to observe the deposition of collagen fibers in the blood vessels.

[0075] Experimental results are as follows Figure 11 As shown, smooth muscle cell-specific overexpression of TRPML1 can significantly reduce the degradation of elastic fibers, the increase in media thickness, and the deposition of collagen fibers in aortic dissection, indicating that smooth muscle cell-specific overexpression of TRPML1 improves the histopathological damage of aortic dissection.

[0076] Experimental Example 5

[0077] A mouse model of Marfan syndrome was established and administered ML-SA5.

[0078] Given that smooth muscle cell-specific overexpression of TRPML1 can improve aortic dissection, we next administered the TRPML1 ion channel agonist ML-SA5 to an aortic dissection model to investigate the effect of ML-SA5 on aortic dissection. The specific steps are as follows:

[0079] (1) Grouping of animals:

[0080] Twenty adult male wild-type mice (denoted as Fbn1) were selected. + / + As the sham-operated group, they were divided into a solvent group and a drug administration group (ML-SA5, 5 mg / kg / 2 d). Twenty adult male Marfan syndrome mice (denoted as Fbn1) were selected. G1039G / + The mice were divided into two groups as the model group: the solvent group and the drug administration group (ML-SA5: 2 mg / kg / 2d; 5 mg / kg / 2d). Six mice per cage were housed under standard conditions (21–24°C, 60% humidity, 12h:12h light / dark cycle) and provided with free access to water and food.

[0081] (2) Detection of the aortic root and ascending aorta diameters in a ML-SA5-treated Marfan syndrome mouse model.

[0082] Small animal ultrasound was used to detect the aortic root and ascending aortic diameter in mice in the Sham group and model group in Example 5.

[0083] Experimental results are as follows Figure 12 The results showed that administration of the TRPML1 agonist ML-SA5 significantly reduced aortic root dilation, indicating that the TRPML1 agonist ML-SA5 improves the progression of aortic dissection.

[0084] (3) Morphological staining method to assess pathological changes of aortic dissection in model mice

[0085] To further evaluate the effect of the TRPML1 agonist ML-SA5 on aortic dissection, the degree of vascular lesions was assessed at the histopathological level using section staining. After the experiments in the Sham group and model group mice in step (1), specimens from the aortic root and ascending aorta were taken for paraffin embedding and sectioning, and histopathological evaluation was performed using staining techniques. EVG staining was used to observe the breakage of elastic fibers and the thickness of the vascular media, and Masson staining was used to observe the deposition of collagen fibers in the blood vessels.

[0086] Experimental results are as follows Figure 13As shown, the TRPML1 agonist ML-SA5 can significantly reduce the degradation of elastic fibers, the increase in media thickness, and the deposition of collagen fibers in aortic dissection, indicating that the TRPML1 agonist ML-SA5 improves the histopathological damage of aortic dissection.

[0087] The above-described embodiments are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope of the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of ML-SA5, a highly specific small molecule agonist of TRPML1, in the preparation of drugs for treating aortic aneurysm or aortic dissection.

2. The application according to claim 1, characterized in that, The aortic aneurysm in question is an abdominal aortic aneurysm.

3. The application according to claim 1, characterized in that, The aortic dissection is Marfan syndrome.

4. The application according to claim 1, characterized in that, The ML-SA5 is administered via intraperitoneal injection.

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