WO2021030978A1 - Use of niclosamide ethanolamine salt in preparation of drugs for treating chemotherapy-realated muscle injury - Google Patents

Abstract

Disclosed is the use of niclosamide ethanolamine salt in the preparation of drugs for treating chemotherapy-realated muscle injury, which relates to a new use of niclosamide ethanolamine salt. Studies have found that niclosamide ethanolamine salt can improve muscle function in model mice with muscle injuries, ameliorate muscle atrophy caused by chemotherapy drugs, increase the body weight of the model mice with muscle injuries, ameliorate pathological muscle injuries of the model mice with muscle injuries, and inhibit p38 MAPK/FoxO3a signaling pathway activation in muscles. According to the above-mentioned studies, it can be concluded that NEN has a relatively strong therapeutic effect on chemotherapy-related muscle injuries, and the functional mechanism thereof is related to the inhibition of the p38 MAPK/FoxO3a signaling pathway.

Description

Application of niclosamide ethanolamine salt in preparing medicine for treating chemotherapy-related muscle injury Technical field

This application relates to a new application of Niclosamide ethanolamine salt (NEN), and more specifically, to the application of Niclosamide ethanolamine salt in the preparation of drugs for the treatment of chemotherapy-related muscle damage.

Background technique

With the increasing incidence of tumors, chemotherapy, as an important means of treating tumors, has become more and more frequent in clinical applications. Muscle damage (including muscle atrophy, functional disuse, etc.) is one of the most common complications during chemotherapy. Studies have shown that muscle volume and muscle function have a great correlation with the prognosis of cancer patients. However, the current treatment methods for chemotherapy-related muscle injuries are extremely limited.

Niclosamide ethanolamine salt has a wide range of biological effects. In recent years, studies have shown that such drugs have anti-tumor, hypoglycemic, and proteinuria effects.

Summary of the invention

The purpose of this application includes providing an application of niclosamide ethanolamine salt in the preparation of a medicine for the treatment of chemotherapy-related muscle injury.

In order to achieve the above objective, the present application provides an application of niclosamide ethanolamine salt in the preparation of a medicine for treating chemotherapy-related muscle injury.

In some embodiments of the present application, the niclosamide ethanolamine salt is used in the preparation of a medicine for the treatment of chemotherapy-related muscle injury.

In some embodiments of the present application, the treatment of chemotherapy-related muscle damage includes improving muscle function, and the improving muscle function includes improving grip and exercise tolerance.

In some embodiments of the present application, the treatment of chemotherapy-related muscle damage includes weight gain.

In some embodiments of the present application, the treatment of chemotherapy-related muscle damage includes improving muscle atrophy.

In some embodiments of the present application, the treatment of chemotherapy-related muscle damage includes improving muscle pathological damage.

In some embodiments of the present application, the niclosamide ethanolamine salt has a therapeutic effect on chemotherapy-related muscle damage and is related to the inhibition of the p38 MAPK/FoxO3a signaling pathway.

In some embodiments of the application, the medicine further includes pharmaceutical excipients.

In some embodiments of the present application, the drug is a tablet, capsule, granule, oral liquid, patch or gel.

In some embodiments of the application, the drug is an oral administration preparation, an injection administration preparation, a mucosal administration preparation or a transdermal administration preparation.

In some embodiments of the present application, the oral administration preparation is a tablet, capsule, granule or oral liquid; the mucosal administration preparation is a patch or gel; the transdermal administration preparation is Patch or gel.

The beneficial effects of this application:

The research of this application found that: Niclosamide ethanolamine salt can improve the muscle function of muscle injury model mice (including improving the grip and exercise tolerance of muscle injury model mice), and improve the muscle atrophy caused by chemotherapy drugs (including improving muscle injury model Muscle capacity and weight of TA, EDL and Gas in mice), increase the body weight of muscle injury model mice, improve muscle pathological damage in muscle injury model mice, and inhibit the activation of the p38 MAPK/FoxO3a signaling pathway in the muscle. According to the above studies, it can be concluded that NEN has a strong therapeutic effect on chemotherapy-related muscle damage (including muscle atrophy, functional disuse, etc.), and its mechanism of action is related to the inhibition of the p38 MAPK/FoxO3a signaling pathway.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present application more clearly, the following will briefly introduce the drawings needed in the embodiments.

Figure 1A and Figure 1B are the results of the effect of NEN on the muscle function of muscle injury model mice; Figure 1A is the effect of NEN on the limb grip of muscle injury model mice, and Figure 1B is the exercise endurance of NEN on muscle injury model mice Among them, the model group compared with the control group, *p<0.05, ***p<0.001; the treatment group compared with the model group, #p<0.05.

Figure 2 shows the effect of NEN on the body weight of muscle injury model mice; among them, the model group and the control group, ***p<0.001; the treatment group and the model group, ##p<0.01, ###p< 0.001.

Figure 3A, Figure 3B, Figure 3C, and Figure 3D show the results of NEN's effect on muscle volume and weight in muscle injury model mice; Figure 3A shows the effect of NEN on the tibialis Anterior (TA) and toe length of muscle injury model mice. Extensor digitorum longus (EDL) and gastrocnemius (Gastrocenmius, GAs) muscle capacity results. Figure 3B, Figure 3C, and Figure 3D show the effect of NEN on the muscle weight of TA, EDL, and GAs in muscle injury model mice. Affect the results; among them, the model group compared with the control group, **p<0.01, ***p<0.001; the treatment group compared with the model group, #p<0.05.

Figure 4A, Figure 4B, Figure 4C and Figure 4D are the results of NEN's effect on muscle pathological damage in muscle injury model mice; Figure 4A is the statistical results of the effect of NEN on muscle fiber cross-sectional area of muscle injury model mice, Figure 4B The statistical results of the effect of NEN on the distribution of muscle fiber cross-sectional area of muscle injury model mice. Figure 4C shows the results of PAS staining showing the effect of NEN on the muscle fiber cross-sectional area of muscle injury model mice. Figure 4D shows NEN. The results of the effect on the ultrastructure of muscle fibers in muscle injury model mice. In Figure 4D, the arrow points to the Z line of the muscle fiber. Among them, the model group compared with the control group, ***p<0.001; the treatment group compared with the model group, ##p<0.01.

Figure 5A, Figure 5B, and Figure 5C are the results of NEN's effect on the p38 MAPK/FoxO3a signaling pathway in the muscles of muscle injury model mice; Figure 5A is the Western blot experiment showing the results of NEN's effect on p38 MAPK/FoxO3a signaling pathway proteins, Figure 5B The statistical results of the effect of NEN on the expression of p-p38 MAPK/p38 MAPK protein in muscle injury model mice. Figure 5C shows the statistical results of the effect of NEN on the expression of FoxO3a protein in muscle injury model mice. Among them, the model group is compared with the control group. * p<0.05, **p<0.01; comparison between treatment group and model group, #p<0.05.

detailed description

The embodiments of the present application will be described in detail below in conjunction with specific examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If specific conditions are not specified in the examples, it shall be carried out according to conventional conditions. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

The following studies were carried out in the embodiments of this application:

1. To study the effect of NEN treatment on the muscle function of chemotherapeutic-induced muscle injury model mice;

2. To study the effect of NEN treatment on the body weight of mice with muscle injury induced by chemotherapy;

3. To study the effect of NEN treatment on muscle atrophy in mice with muscle injury induced by chemotherapy;

4. To study the effect of NEN treatment on muscle pathological damage in mice with chemotherapeutic drug-induced muscle injury;

5. To study the effect of NEN treatment on the activation of p38MAPK/FoxO3a signaling pathway in muscles of chemotherapeutic-induced muscle injury model mice.

1. Experimental method

1. Animal model

Male BALB/c mice (20-25g) were purchased from the Medical Laboratory Animal Center of Southern Medical University. Animal experiments are carried out in strict accordance with the animal ethics guidelines and regulations of Guangzhou University of Traditional Chinese Medicine. The experimental animals were controlled under the conditions of a constant room temperature of 20±1°C, 12 hours of light and 12 hours of dark cycles, while freely eating and drinking. The chemotherapeutic-induced muscle injury model mice were induced by a one-time tail vein injection of 10.4 mg/kg doxorubicin (purchased from Sigma, USA).

The experimental mice were randomly assigned to the following groups (6 in each group): normal control group (ie, the control group in the drawings of this application), muscle injury model group (ie, the model group in the drawings of this application), and NEN treatment group (That is, the treatment group in the attached drawings of this application), wherein the mice in the control group are normal mice and fed regular food; the mice in the model group are muscle injury model mice and are fed regular food; the mice in the treatment group are muscle injury model mice The rats were fed with food supplemented with NEN on the basis of regular food. NEN was purchased from China's Hubei Shengtian Hengchuang Biotechnology Co., Ltd., and added to the diet of mice in the treatment group at a standard ratio of 2g/kg. The above experimental treatment lasted 2 weeks.

2. Grasping power and exercise tolerance test

The mouse grip (Newton N) was measured using a grip tester (China Anhui Zhenghua Biological Instrument Equipment Co., Ltd.). An animal experiment treadmill (China Anhui Zhenghua Bio-Instrument Equipment Co., Ltd.) was used to determine the number of electric shocks (times/m/s, Shocks/Mile/s) of mice within a specified time and distance.

3. Organizational preparation

After the mice were sacrificed, the muscle tissue TA, EDL, and GAs were immediately separated, and the water was sucked dry with a paper towel, and then weighed. Among them, TA was fixed with 10% formalin for PAS staining, EDL was fixed with 2.5% glutaraldehyde for electron microscopy, and GAs were immediately frozen in liquid nitrogen and stored at -80°C for subsequent experimental studies.

4. Pathological examination

TA paraffin sections (3μm thick) were stained with PAS to evaluate the cross-sectional area of muscle. The muscle fiber area statistics were processed and analyzed using ImageJ software (National Institutes of Health). The long axis section of EDL is used for electron microscopy.

5. Western blot

Homogenize the frozen muscle (GAs) tissue in the lysis buffer, balance the total protein concentration, and separate it by SDS-PAGE gel electrophoresis, and transfer the protein to PVDF (polyvinylidene fluoride) membrane. Put the PVDF membrane in TBS buffer containing 0.5g/l skimmed milk powder, react at room temperature for 1 hour, block non-specific sites on the membrane, then add the primary antibody, and incubate overnight at 4°C with shaking. After washing, ChemiDocTM MP imaging system (Bio-Rad, USA) was used to detect and analyze protein bands. The results were compared using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal reference. The antibodies of p-p38 MAPK, p38 MAPK, and FoxO3a were purchased from CST, USA; GAPDH antibody was purchased from Proteintech, USA.

6. Statistical methods

The measurement data are expressed as mean ± standard deviation. The statistical difference between the two groups of samples was analyzed by independent sample t test, the comparison between multiple groups of samples was performed by one-way analysis of variance, and the statistical analysis was processed by SPSS16.0 statistical software. When P<0.05, the difference is considered to be statistically significant.

2. Results

1. NEN can improve the muscle function of muscle injury model mice

Figures 1A and 1B show the results of NEN's ability to improve muscle function in muscle injury model mice. Figure 1A shows the effect of NEN on the holding power of muscle injury model mice, and Figure 1B shows the effect of NEN on the exercise tolerance of muscle injury model mice. In the control group, model group and treatment group, each group has 6 mice, as shown in Figure 1A and Figure 1B. Compared with the control group mice, the model group mice's grip and exercise tolerance are significantly reduced; After 2 weeks of NEN, compared with mice in the model group, the grip and exercise tolerance of the mice in the treatment group were significantly improved.

2. NEN can increase the weight of muscle injury model mice

Figure 2 shows the effect of NEN on the body weight of muscle injury model mice. In the control group, model group, and treatment group, each group has 6 mice. As shown in Figure 2, the weight of the model group mice is significantly reduced compared with the control group mice; after 2 weeks of NEN treatment, Compared with mice in the model group, the weight of the mice in the treatment group increased significantly.

3. NEN can improve muscle wasting caused by chemotherapy drugs

Figure 3A, Figure 3B, Figure 3C and Figure 3D show the effect of NEN on muscle atrophy in muscle injury model mice. Figure 3A shows the effect of NEN on the muscle capacity of TA, EDL and Gas in muscle injury model mice. Figure 3B shows the effect of NEN on the TA muscle weight of muscle injury model mice. Figure 3C shows the effect of NEN on muscle injury model mice. The results of the effect of the EDL muscle weight of the mouse, Figure 3D shows the effect of the NEN on the muscle weight of the GAs of the muscle injury model mice. In the control group, model group and treatment group, each group has 6 mice. Compared with the control group, the TA, EDL and GAs of the model group were significantly atrophy (muscle volume and muscle weight were significantly reduced); After 2 weeks of NEN treatment, the muscle volume of TA, EDL and GAs in the treatment group was significantly higher than that of the model group, and the muscle weight was also significantly higher than that of the model group.

4. NEN can improve muscle pathological damage caused by chemotherapy drugs

Fig. 4A, Fig. 4B, Fig. 4C and Fig. 4D are the results of the effect of NEN on the muscle pathological damage of the muscle injury model mice; Fig. 4A is the results of the effect of NEN on the muscle fiber cross-sectional area of the muscle injury model mice, and Fig. 4B is The effect of NEN on the distribution of muscle fiber cross-sectional area of muscle injury model mice. Figure 4C shows the results of PAS staining showing the effect of NEN on the muscle fiber cross-sectional area of muscle injury model mice. Figure 4D shows the effect of NEN on muscle injury model mice. The effect of the ultrastructure of the muscle fibers in mice results. In the control group, the model group and the treatment group, each group has 6 mice, as shown in Figure 4A and Figure 4C. Compared with the control group, the muscle fiber cross-sectional area of the model group is significantly smaller; After 2 weeks of NEN treatment, the cross-sectional area of the muscle fibers of the mice in the treatment group became significantly larger; as shown in Figure 4B, the cross-sectional area of the muscle fibers of the mice in the model group was overall smaller, and the frequency distribution graph showed that the overall cross-sectional area was shifted to the left. The distribution of mice in the treatment group is similar to that of the control group; as shown in Figure 4C, the Z-line in the muscle fibers is significantly thinner than that in the control group. After NEN treatment , The Z-line of the treatment group mice is obviously thicker than that of the model group.

5. NEN can inhibit the activation of p38 MAPK/FoxO3a signaling pathway

Figure 5A, Figure 5B and Figure 5C are the results of NEN's effect on the p38 MAPK/FoxO3a signaling pathway in the muscles of muscle injury model mice. Figure 5A is the Western blot experiment showing the results of NEN's effect on p38 MAPK/FoxO3a signaling pathway proteins. 5B is the statistical result of the influence of NEN on p-p38 MAPK protein, and Figure 5C is the statistical result of the influence of NEN on FoxO3a protein.

p-p38 MAPK is the phosphorylated form of p38 MAPK, that is, the molecular form of p38 MAPK in an activated state. The activation of FoxO3a protein will initiate a series of signaling pathways leading to muscle atrophy, which in turn leads to muscle disuse and decreased muscle function (shown as decreased grip and exercise endurance). p38MAPK is a regulatory protein with a wide range of biological effects, located upstream of FoxO3a protein. After p38MAPK is activated to p-p38 MAPK, p-p38 MAPK can induce the activation of downstream FoxO3a protein, which may stimulate the downstream signaling pathway of FoxO3a protein.

It can be seen from Figure 5A that the expression levels of p38 MAPK in the control group, model group, and treatment group are almost the same, while the content of p-p38 MAPK is different. GAPDH is used as an internal reference in the control group and model group. The expression levels in the mice and the mice in the treatment group are basically the same. Therefore, the ordinate (p-p38MAPK/p38 MAPK) in Figure 5B represents the activation ratio of p38 MAPK in mice, and the ordinate in Figure 5C (FoxO3a/ GAPDH) represents the expression level of FoxO3a protein in mice.

In the control group, model group, and treatment group, each group has 6 mice. From Figure 5A to Figure 5C, it can be seen that compared with the control group, the p-p38MAPK/p38 MAPK in the muscle of the model group And FoxO3a protein increased significantly. After NEN treatment, the p-p38MAPK/p38 MAPK and FoxO3a protein of the mice in the treatment group were significantly reduced compared with the model group. In other words, NEN has an inhibitory effect on the p38 MAPK/FoxO3a signaling pathway.

The research results of this application show that after the use of NEN to treat muscle injury model mice, the muscle strength, weight, and pathological damage of muscle injury model mice have significantly improved indicators used to measure muscle atrophy and muscle function. The p38MAPK/FoxO3a signaling pathways related to sleep and muscle disuse are inhibited. Therefore, it is inferred that NEN plays its role in improving muscle atrophy muscle function by inhibiting the p38 MAPK/FoxO3a signaling pathway.

The above results indicate that as a drug with a wide range of biological effects, NEN can improve the muscle function of muscle injury model mice (including improving the grip and exercise tolerance of muscle injury model mice), and improve the muscle atrophy caused by chemotherapy drugs (including enhancing Muscle capacity and weight of TA, EDL and Gas in muscle injury model mice), increase the body weight of muscle injury model mice, improve muscle pathological damage in muscle injury model mice, and inhibit the activation of the p38 MAPK/FoxO3a signaling pathway in the muscle. In summary, NEN has a strong therapeutic effect on chemotherapy-related muscle injuries (including muscle atrophy, functional disuse, etc.).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the application, not to limit them; although the application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features; these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the application range.

In addition, those skilled in the art can understand that although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments means that it is within the scope of the present application And form different embodiments. For example, in the above claims, any one of the claimed embodiments can be used in any combination. The information disclosed in the background technology section is only intended to deepen the understanding of the overall background technology of the application, and should not be regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art known to those skilled in the art.

Claims (10)

1. Application of niclosamide ethanolamine salt in the preparation of medicines for treating chemotherapy-related muscle injuries.
2. The application of claim 1, wherein the treatment of chemotherapy-related muscle damage includes improving muscle function, and the improving muscle function includes improving grip and improving exercise tolerance.
3. The application of claim 1, wherein the treatment of chemotherapy-related muscle damage includes weight gain.
4. The application of claim 1, wherein the treatment of chemotherapy-related muscle damage includes improving muscle atrophy.
5. The application according to claim 1, wherein the treatment of chemotherapy-related muscle damage includes improving muscle pathological damage.
6. The use according to claim 1, wherein the niclosamide ethanolamine salt has a therapeutic effect on chemotherapy-related muscle damage and is related to the inhibition of the p38MAPK/FoxO3a signaling pathway.
7. The application according to claim 1, wherein the medicine further comprises pharmaceutical excipients.
8. The application according to claim 1, wherein the medicine is a tablet, capsule, granule, oral liquid, patch or gel.
9. The application according to claim 1, wherein the medicine is an oral administration preparation, an injection administration preparation, a mucosal administration preparation or a transdermal administration preparation.
10. The application according to claim 9, wherein the oral administration preparation is a tablet, capsule, granule or oral liquid; the mucosal administration preparation is a patch or gel; the transdermal preparation The administration preparation is a patch or a gel.

Images