Use of acidic sphingomyelinase and inhibitors thereof for the preparation of a medicament for the treatment of inflammatory myopathies

Abstract

The application discloses application of acid sphingomyelinase and an inhibitor thereof in preparation of a medicine for treating inflammatory myopathy. It is found in the application that knocking out or inhibiting acid sphingomyelinase can effectively improve disease indexes of an animal model of inflammatory myopathy and effectively relieve inflammatory myopathy. Therefore, acid sphingomyelinase inhibitors including amitriptyline have the prospect of being developed into a medicine for treating inflammatory myopathy.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of acid sphingomyelinase and its inhibitors in the preparation of drugs for treating inflammatory myopathy. Background Technology

[0002] Sphingomyelinase (SMase) is a glycoprotein that hydrolyzes sphingomyelin (SM). In mammals, it exists in three forms depending on the pH environment: acidic sphingomyelinase (ASM), neutral sphingomyelinase (NSM), and alkaline sphingomyelinase (Alk-SM). Among them, ASM accounts for 90% of the total biological activity of SMase and plays an important role in the production of ceramides in the body.

[0003] Inflammatory myopathy is a heterogeneous immune disease characterized by skeletal muscle involvement. It involves a variety of autoantibodies and can be classified into several clinical subtypes based on the different antibodies, including polymyositis, dermatomyositis, inclusion body myositis, immune-mediated necrotizing myopathy, juvenile idiopathic myositis, and antisynthetic enzyme antibody syndrome.

[0004] This invention is proposed to develop applications of acid sphingomyelinase and its inhibitors in the treatment of inflammatory myopathy. Summary of the Invention

[0005] The purpose of this invention is to provide the use of acid sphingomyelinase and its inhibitors in the preparation of medicaments for treating inflammatory myopathy.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] Application of acid sphingomyelinase and its inhibitors in the preparation of drugs for treating inflammatory myopathy.

[0008] Furthermore, the drug uses an inhibitor of acid sphingomyelinase as its active ingredient and also contains a pharmaceutically acceptable carrier or excipient, and is formulated into a pharmaceutically acceptable dosage form.

[0009] Furthermore, the carrier or auxiliary material is a solid, liquid, or semi-solid.

[0010] Furthermore, the dosage forms include tablets, capsules, and injections.

[0011] Furthermore, the inhibitor is amitriptyline.

[0012] Beneficial effects:

[0013] This invention reveals that knockout or inhibition of acid sphingomyelinase can effectively improve disease markers in animal models of inflammatory myopathy and effectively alleviate the condition. Therefore, acid sphingomyelinase inhibitors, including amitriptyline, show promise as drugs for the treatment of inflammatory myopathy. Attached Figure Description

[0014] Figure 1 To construct an EAM model in ASM gene knockout mice; (A) muscle strength, (B) serum CK level, (C) gross image of mouse spleen, (D) spleen weight, (E) H&E staining of mouse muscle tissue sections, and (F) quantitative score of H&E staining. * indicates P<0.05, ** indicates P<0.1, and *** indicates P<0.01.

[0015] Figure 2 To demonstrate the effectiveness of AMI treatment in an EAM mouse model. (A) Muscle strength (B) Serum CK level (C) Gross image of mouse spleen (D) Spleen weight (E) H&E staining of mouse muscle tissue sections (F) Quantitative H&E staining score. *<0.05, * indicates P<0.05, ** indicates P<0.1, *** indicates P<0.01. Detailed Implementation

[0016] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0017] I. Experimental Materials

[0018] Amitriptyline: HY-B0527A, MCE;

[0019] Pertussis toxin: GC17532, GLPBIO;

[0020] Environmentally friendly GD fixative: G1111-100ML, Servicebio;

[0021] Incomplete Freund's Adjuvant: 7002, Chondrex;

[0022] Complete Freund's Adjuvant: 7001, Chondrex;

[0023] Disposable syringe: 1ml, 0.45*16 (brown), Xinjin Shifeng.

[0024] II. Experimental Methods

[0025] 1. Establish a mouse model of polymyositis (EAM model).

[0026] Six- to eight-week-old female C57BL / 6 mice were immunized subcutaneously in the left hind limb on days 1 and 7 with 1.5 mg of guinea pig myosin emulsified with an equal volume of complete Freund's adjuvant containing 5 mg / mL of Mycobacterium tuberculosis. Immediately after each immunization, mice were intraperitoneally injected with 500 ng of pertussis toxin prepared with 200 μl of physiological saline. Booster immunizations were administered twice weekly via subcutaneous emulsion of incomplete Freund's adjuvant at the tail base and left ventricular region. The control group received two doses of pertussis toxin emulsion of physiological saline and complete Freund's adjuvant, followed by booster immunizations with emulsion of physiological saline and incomplete Freund's adjuvant. Mice were sacrificed on day 14 after the first immunization, and blood, spleen, and muscle tissue were collected.

[0027] 2. Muscle strength test

[0028] The grip strength of mice and rats was measured using the YLS-13A rat and rat grip strength tester (Jinan Yiyan Technology Development Co., Ltd.) before and at the end of the animal experiments. Each mouse was tested 3 times and the average value was taken.

[0029] 3. Pathological score of muscle tissue H&E staining

[0030] Fresh muscle tissue was fixed in muscle tissue fixative for 24 hours, then embedded in paraffin and sectioned. After H&E staining, the inflammatory cell infiltration was scored according to the following criteria: no inflammatory cell infiltration was 0 points, less than 5 muscle fibers were infiltrated by inflammatory cells, 5-30 muscle fibers were infiltrated by 2 points, the entire muscle bundle was involved by 3 points, and extensive infiltration was 4 points. If multiple lesions appeared on one section, an additional 0.5 points were added.

[0031] 4. Serum muscle enzyme levels

[0032] After blood collection, the mice were euthanized by decapitation, and whole blood was collected to obtain serum. The serum was then sent to Wuhan Saiwell Biotechnology Co., Ltd. for detection of myokinase CK using a fully automated biochemical analyzer (Shenzhen Leidu Life Science Technology).

[0033] 5. Weighing and photographing the spleen

[0034] After blood was drawn, the mice were euthanized by decapitation, and their spleens were removed, weighed, and photographed.

[0035] 6. Data Processing

[0036] Data were plotted using Prism6 software, and analyzed using SPSS software. One-way ANOVA and Bonferroni analysis were used to analyze differences between groups and pairwise comparisons. P < 0.05 was considered statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0037] III. Experimental Results

[0038] 1. ASM participates in the EAM model

[0039] In mice with the gene encoding acid sphingomyelinase (ASM) knocked out (SMPD1), - / - EAM model was constructed in mice, and the results showed that it was similar to wild-type model mice (SMPD1). + / + Compared to SMPD1 - / - In mice, muscle strength was increased, serum CK levels were decreased, spleen weight was reduced, inflammatory cell infiltration in the muscle was reduced, and pathological scores were alleviated, such as... Figure 1 .

[0040] 2. AMI effectively treats EAM mouse model

[0041] EAM model was established in wild-type mice and treated with the ASM inhibitor amitriptyline (AMI) at 1 mg / kg and 10 mg / kg. Results showed that, compared with the model group, AMI treatment improved muscle strength, reduced serum CK levels, decreased spleen weight, reduced inflammatory cell infiltration in muscle, and reduced pathological scores. Figure 2 .

[0042] The above experimental results indicate that knockout or inhibition of acid sphingomyelinase can effectively improve disease indicators in animal models of inflammatory myopathy and effectively alleviate the disease. Therefore, acid sphingomyelinase inhibitors, including amitriptyline, show promise as drugs for the treatment of inflammatory myopathy.

[0043] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. The use of amitriptyline in the preparation of a drug for treating inflammatory myopathy, wherein the inflammatory myopathy is polymyositis.

2. The application according to claim 1, characterized in that: The drug uses amitriptyline as its active ingredient and also contains pharmaceutically acceptable excipients, and is formulated into a pharmaceutically acceptable dosage form.

3. The application according to claim 2, characterized in that: The excipients are solid, liquid, or semi-solid excipients.

4. The application according to claim 2, characterized in that: The dosage form is tablet, capsule, or injection.

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