CN112807309A

CN112807309A - Application of TRPML1 specific small molecule activator ML-SA5

Info

Publication number: CN112807309A
Application number: CN202110108815.1A
Authority: CN
Inventors: 王午阳; 陈蓉
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-05-18

Abstract

The invention discloses an application of a TRPML1 specific small molecule activator ML-SA5 and an application of a TRPML1 specific small molecule activator ML-SA5 in the aspect of preventing and treating ulcerative colitis. The invention has the beneficial effects that: the TRPML1 specific small molecule activator ML-SA5 can improve the damage of intestinal mucosa tissues, provides a new medicine for preventing and treating ulcerative colitis, and has good market value and clinical application prospect.

Description

Application of TRPML1 specific small molecule activator ML-SA5

Technical Field

The invention relates to the technical field of medicines, in particular to application of a TRPML1 specific small-molecule activator ML-SA5 in the aspect of preventing and treating ulcerative colitis.

Background

Ulcerative Colitis (UC) is a chronic nonspecific inflammatory disease of gastrointestinal tract with unknown etiology, and the pathological changes can affect the mucous membrane of colon and even the whole gastrointestinal tract. Clinically, patients may have repeated abdominal pain, diarrhea, mucous bloody stool, and even various systemic complications such as blurred vision, arthralgia, and rash. The pathological changes mainly take intestinal mucosal immune response abnormality as the main factor, and the occurrence mechanism may be: under the participation of intestinal flora, environmental factors act on genetically susceptible patients to start intestinal innate and acquired immune response, which leads to pathological changes such as intestinal mucosa barrier damage, inflammatory hyperplasia, ulcer disunion and the like, and the environmental, infection, immunity and genetic factors are closely related to the UC pathogenesis.

Disclosure of Invention

The main purpose of the present application is to provide a new use of TRPML 1-specific small molecule activator ML-SA5 for prevention and treatment of ulcerative colitis, so as to solve the problems in the related art.

In order to achieve the above purpose, the invention provides the following technical scheme:

the application of the TRPML1 specific small molecule activator ML-SA5 and the application of the TRPML1 specific small molecule activator ML-SA5 in preventing and treating ulcerative colitis.

The application of the TRPML1 specific small molecule activator ML-SA5 is the application of the TRPML1 specific small molecule activator ML-SA5 in the aspect of medicines for preventing ulcerative colitis as a preferred embodiment.

The application of the TRPML1 specific small molecule activator ML-SA5 is the application of the TRPML1 specific small molecule activator ML-SA5 in the aspect of medicines for treating ulcerative colitis as a preferred embodiment.

As a preferred embodiment, the effective dosage of the TRPML1 specific small molecule activator ML-SA5 is 0.3mg/kg-1.8 mg/kg.

The application of the TRPML1 specific small molecule activator ML-SA5 is that as a preferred embodiment, the effective dose of the TRPML1 specific small molecule activator ML-SA5 is 0.3 mg/kg.

The application of the TRPML1 specific small molecule activator ML-SA5 is that as a preferred embodiment, the effective dose of the TRPML1 specific small molecule activator ML-SA5 is 0.9 mg/kg.

The application of the TRPML1 specific small molecule activator ML-SA5 is that as a preferred embodiment, the effective dose of the TRPML1 specific small molecule activator ML-SA5 is 1.8 mg/kg.

The invention has the beneficial effects that: the TRPML1 specific small molecule activator ML-SA5 can improve the damage of intestinal mucosa tissues, provides a new medicine for preventing and treating ulcerative colitis, and has good market value and clinical application prospect.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:

FIG. 1 is a chemical structural diagram of TRPML1 specific small molecule activator ML-SA5 according to the present invention;

FIG. 2 is a graph of the time pattern of preparation and administration of DSS-induced ulcerative colitis model mice;

FIG. 3 shows the results of body weight improvement in mice with ulcerative colitis induced by combination administration of ML-SA5 with DSS;

FIG. 4 is a graph showing the improvement of colon necrosis caused by ML-SA5 in combination with DSS-induced ulcerative colitis;

FIG. 5 is a graph of the disease activity index improvement results of ML-SA5 combination for DSS-induced ulcerative colitis mice;

FIG. 6 shows that ML-SA5 combined administration significantly improved the degree of inflammatory cell infiltration and mucosal destruction in colon tissue of mice with DSS-induced ulcerative colitis.

Detailed Description

In order to make the technical solutions in the embodiments of the present application better understood, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

The application discloses a new application of a TRPML1 specific small molecule activator ML-SA5 and application of the activator in preventing and treating ulcerative colitis. In particular to the application of the activator for preventing and treating ulcerative colitis, the activator can improve the damage of intestinal mucosa.

When the activator is used for preventing and treating ulcerative colitis, the effective dose is 0.3mg/kg-1.8mg/kg, preferably the effective dose is 0.3mg/kg, and more preferably the effective dose is 0.9 mg/kg; further preferably, the effective dose is 1.8 mg/kg.

Our previous studies indicate that the lysosomal cation channel TRPML1 is a specific target for the regulation of autophagy, and that autophagy activity can be specifically regulated by altering the activity of the TRPML1 channel using agonists or inhibitors of TRPML 1. Therefore, the following is an experiment of the present application to demonstrate that the specific small molecule activator of TRPML1 ML-SA5 (see fig. 1 for its chemical structure) has preventive and therapeutic effects on ulcerative colitis.

1. Test method

A DSS-induced ulcerative colitis model was prepared using C57BL/6J mice and divided into 3 groups, namely a normal group, a DSS-induced colitis model group, a DSS-making model group combined with a low dose ML-SA5(0.3mg/kg) group, a medium dose ML-SA5(0.9mg/kg) group and a high dose ML-SA5(1.8mg/kg) group.

UC modeling, preparation of ML-SA5 intervention and time pattern of administration, as shown in figure 2:

three days before the beginning of the establishment of the ulcerative colitis model, all the C57BL/6J experimental mice freely drink distilled water, and at the same time point every day, ML-SA5 intervention group mice are respectively injected with different doses of ML-SA5 liquid with the same volume by intraperitoneal injection, while normal group and DSS model group are injected with normal saline with the same volume by intraperitoneal injection to serve as a control. And then, making a model by utilizing the DSS for seven days, wherein the normal group continues to freely drink distilled water, the other four groups start to freely drink distilled water dissolved with 3% DSS, the newly prepared DSS solution is replaced every other two days, and the intraperitoneal administration mode is the same as that of the previous three days (the normal group and the DSS model group are subjected to intraperitoneal injection of physiological saline, and the drug intervention group is subjected to intraperitoneal injection of ML-SA5 solution with different doses). During the molding administration period, observing the state of the mouse every day, weighing the weight of the mouse, observing the characteristics of the excrement of the mouse, detecting the hematochezia condition of the excrement of the mouse, and recording the result; on day 7 of molding, after the relevant index was determined, the colon tissue of the mouse was taken out, the length of the colon was measured and the results of pathological analysis were carried out.

2. Test results

2.1 methods for measuring weight change: the body weights of the mice were measured daily on days 0 to 7 after administration of DSS, the body weights on day 0 were used as the basic body weight, the body weight ratios on the corresponding days were calculated, and the body weight change curves were made using graphpad7.0 software, and the results are shown in fig. 3:

as can be seen from fig. 3: the combination of ML-SA5(0.3, 0.9 and 1.8mg/kg) with different doses obviously improves the condition of rapid weight loss of mice caused by DSS-induced ulcerative colitis, and the effect of 0.3mg/kgML-SA5 is the best.

2.2 measurement of Colon Length

On day 7 after molding, the colon of the mouse was removed, laid flat on gauze soaked with 0.9% physiological saline, stretched gently along the long axis, and the length from the ileocecal portion to the anal end was measured with a ruler, as shown in fig. 4. The length of the colon may reflect the degree of necrosis of the colon, the more severe the necrosis, the shorter the length of the colon.

As can be seen from fig. 4: the presented ML-SA5 colon length in the dried group (0.3mg/kg) was significantly longer than in the dried group. The statistical chart compares the colon length of a control group, a DSS group and a DSS group combined with different doses of ML-SA5(0.3, 0.9 and 1.8mg/kg), and suggests that the ML-SA5 intervention can obviously improve the colon necrosis condition of mice caused by DSS-induced ulcerative colitis, and the effect is best at 0.3mg/kgML-SA 5.

2.3 evaluation method and results of Disease Activity Index (DAI)

During the molding administration period, the change condition of the body weight of the mouse is observed every day, the stool character and the hematochezia condition are recorded at the same time, the scoring is carried out according to the scoring standard shown in the table I, and the DAI of the mouse is the sum of the weight loss percentage, the stool character and the stool occult blood condition.

TABLE 1 Disease Activity Index (DAI) score criteria

Scoring	Weight loss (%)	Stool characteristics	Condition of hematochezia
				0	0	Is normal	Normal bloodless
1	1-5％	Soft and good in formation	Occult blood (-)
				2	6-10％	Soft and unable to form feces	Occult blood (+)
3	11-18％	Is very soft	Blood spots in the rectum
				4	＞18％	Watery diarrhea	Hematochezia of rectum

FIG. 5 shows statistics of Disease Activity Index (DAI) of the control group, DSS group, and DSS group combined with different doses of ML-SA5(0.3, 0.9, 1.8mg/kg) from day one to day seven, as can be seen from FIG. 5: the ML-SA5 intervention can obviously improve the disease activity index caused by DSS-induced ulcerative colitis.

2.4 methods and results of Colon histopathology analysis and evaluation

On day 7 after molding, the colons of the mice were removed, observed for colons tissue damage, and scored according to the scoring system described in table 2.

TABLE 2 DSS-induced colitis pathological tissue score criteria

Scoring	Tissue damage	Infiltration of lamina propria inflammatory cells
			0	Is free of	Rare
1	Isolated focal epithelial lesions	Increase, neutrophil infiltration
			2	Mucosal erosion and ulcer	Inflammatory cell mass under the mucosa
3	Extensive deep intestinal wall injury	Transmural inflammatory cell infiltration

The intestinal lumen was then flushed with 0.9% saline. Fixing distal colon tissues at the same position in 4% paraformaldehyde solution, sequentially trimming, dehydrating, transparentizing, waxing, embedding, slicing and the like to prepare paraffin sections, putting the prepared paraffin sections into dimethylbenzene for dewaxing, taking out after 30min, and sequentially transferring the paraffin sections into 100%, 95%, 90%, 80% and 70% ethanol solutions for 5min each time; finally, the mixture was transferred to distilled water and left for 5 min. And then putting the dewaxed section into hematoxylin staining solution, standing for 3min, washing for 1min, transferring into 95% ethanol, and staining with eosin staining solution for 1 min. After dyeing, the sections are transferred to 70%, 80%, 90%, 95% and 100% ethanol solution, dehydrated, then transferred to xylene for 5min, and finally sealed with neutral glue. The pathologist was asked to observe under a light microscope and to perform scoring according to the scoring method described in table 2.

As can be seen from fig. 6: HE staining showed that the level of injury to the colonic mucosal tissue in the ML-SA5 dosed group was significantly lower than the sham group without ML-SA5 intervention.

The above experimental results prove that: the ML-SA5 small molecular compound can obviously improve the intestinal mucosal tissue injury of an ulcerative colitis model mouse. The invention provides a new medicine for preventing and treating ulcerative colitis, and has good market value and clinical application prospect.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

Use of a TRPML 1-specific small molecule activator ML-SA5, characterized in that the TRPML 1-specific small molecule activator ML-SA5 is used for the prevention and treatment of ulcerative colitis.
2. The use of a TRPML 1-specific small molecule activator ML-SA5 according to claim 1, characterized by the use of TRPML 1-specific small molecule activator ML-SA5 in a medicament for the prevention of ulcerative colitis.
3. The use of a TRPML 1-specific small molecule activator ML-SA5 according to claim 1, characterized by the use of TRPML 1-specific small molecule activator ML-SA5 in a medicament for the treatment of ulcerative colitis.
4. The use of a TRPML1 specific small molecule activator ML-SA5 according to claim 1 wherein the effective dose of TRPML1 specific small molecule activator ML-SA5 is 0.3mg/kg to 1.8 mg/kg.
5. The use of a TRPML 1-specific small molecule activator ML-SA5 as claimed in claim 4 wherein the effective dose of the TRPML 1-specific small molecule activator ML-SA5 is 0.3 mg/kg.
6. The use of a TRPML 1-specific small molecule activator ML-SA5 as claimed in claim 4 wherein the effective dose of the TRPML 1-specific small molecule activator ML-SA5 is 0.9 mg/kg.
7. The use of a TRPML 1-specific small molecule activator ML-SA5 as claimed in claim 4 wherein the effective dose of the TRPML 1-specific small molecule activator ML-SA5 is 1.8 mg/kg.