Use of ligustrazine nitrone derivative in preparation of drug for preventing or treating sarcopenia

Abstract

The present invention provides use of a ligustrazine nitrone derivative in the preparation of a drug for preventing or treating sarcopenia. The ligustrazine nitrone derivative of the present invention can effectively improve the muscle mass, muscle strength, and muscle functions of sarcopenia model animals, and has the potential to be developed into the drug for preventing or treating sarcopenia. The ligustrazine nitrone derivative has a structure represented by general formula (I) below.

Description

Application of ligustrazine nitrone derivatives in preparing drugs for preventing or treating sarcopenia Technical Field

[0001] The present invention relates to a new use of ligustrazine nitrone derivatives or pharmaceutically acceptable salts thereof, and particularly relates to the use of ligustrazine nitrone derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for preventing or treating sarcopenia. Background Art

[0002] Sarcopenia (also known as muscle loss) refers to a syndrome characterized by age-related loss of muscle mass, decreased strength, and / or decreased physical function. As muscles weaken, the ability to move decreases, leading to slow movement, poor balance, and easy falls. Epidemiological surveys have found that the incidence of sarcopenia increases year by year with age, and the incidence rises sharply after the age of 50. The prevalence of sarcopenia in people aged ≥65 years is 15%, while it is as high as 50% in people aged ≥80 years (Cohen S, Nathan JA, Goldberg AL. Muscle wasting in disease: molecular mechanisms and promising therapies [J]. Nat Rev Drug Discov, 2015, 14(1): 58-74). Sarcopenia is characterized by high incidence, complex etiology, insidious progression, and widespread adverse effects. However, current research at home and abroad has not yet fully revealed its pathogenesis, and there is no specific drug. Clinically, it is mainly nutritional intervention combined with resistance exercise. However, resistance exercise is difficult to implement for most elderly people, especially those in nursing homes and hospitals. To meet the needs of clinical treatment, the development of therapeutic drugs for sarcopenia is urgent.

[0003] Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention aims to provide a use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in preventing or treating sarcopenia and preparing corresponding drugs.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] The present invention provides a method for preventing or treating sarcopenia using a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof, which comprises administering a therapeutically effective amount of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof to a patient in need.

[0007] The present invention provides use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating sarcopenia.

[0008] The ligustrazine nitrone derivative of the present invention has a structure represented by the following general formula (I):

[0009] in:

[0010] R1 is hydrogen, methyl or

[0011] R2 and R3 are the same or different and are independently selected from hydrogen or C1-C6 alkyl; R4 is sec-butyl, isobutyl, tert-butyl, cyclopentyl or cyclohexyl, and R5 is sec-butyl, isobutyl, tert-butyl, cyclopentyl or cyclohexyl.

[0012] Furthermore, in the present invention, R2 and R3 are the same or different and are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, preferably methyl, ethyl or propyl.

[0013] Furthermore, the ligustrazine nitrone derivative of the present invention is TBN or TN-2:

[0014] According to one embodiment of the present invention, the sarcopenia is primary sarcopenia or secondary sarcopenia. Primary sarcopenia is mainly related to age. Secondary sarcopenia is mainly related to diseases (such as metabolic diseases, chronic heart, lung, liver, kidney diseases, cerebrovascular diseases), drug abuse, reduced physical activity, unhealthy lifestyle, etc.

[0015] According to one embodiment of the present invention, the present invention provides the use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for reducing sarcopenia muscle mass loss, maintaining or increasing muscle mass.

[0016] According to one embodiment of the present invention, the present invention provides use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving muscle strength in sarcopenia.

[0017] According to one embodiment of the present invention, the present invention provides use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving physical function in sarcopenia.

[0018] According to one embodiment of the present invention, the present invention provides use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing muscle fibrosis in sarcopenia.

[0019] According to one embodiment of the present invention, the pharmaceutically acceptable salts of the present invention may be salts formed with inorganic acids, such as salts formed with the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; or salts formed with organic acids, such as salts formed with the following organic acids: methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, hexanoic acid, benzoic acid, salicylic acid, cinnamic acid, cyclopentanepropionic acid, dodecylsulfuric acid, 2-naphthalenesulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, D-gluconic acid, aspartic acid.

[0020] The ligustrazine nitrone derivatives of the present invention or their pharmaceutically acceptable salts can be administered orally or parenterally, for example, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally or subcutaneously. Dosage forms for oral administration may include tablets, pills, soft or hard capsules, granules, powders, micropowders, liquids, emulsions or micropills, but are not limited thereto. Dosage forms for parenteral administration may include eye drops, injections, medicinal drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays, but are not limited thereto.

[0021] The dosage of the ligustrazine nitrone derivative or its pharmaceutically acceptable salt according to the present invention can be 10-5000 mg / person / time according to the dosage of the ligustrazine nitrone derivative. The specific dosage can be different according to the age, sex and weight of the subject, the specific pathological condition and its severity, the route of administration or the diagnosis. The dosage based on the above factors can be determined according to the level of those skilled in the art. The specific mode of administration and the number of administrations can be in accordance with the conventional mode of administration of the ligustrazine nitrone derivative (such as TBN or TN-2), for example, it can be administered once or multiple times daily.

[0022] The present invention provides the use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating sarcopenia. The present invention experimentally investigated the ligustrazine nitrone derivative TBN in a D-galactose-induced sarcopenia mouse model and found that the ligustrazine nitrone derivative TBN can increase the wet weight ratio of the gastrocnemius muscle and effectively reduce the degree of gastrocnemius fibrosis in the sarcopenia model mice. In addition, the ligustrazine nitrone derivative TBN can significantly prolong the climbing time of D-galactose-induced mice, increase the mouse grip and reduce the time it takes to fall from the rotating rod, and significantly improve the muscle strength of the sarcopenic mice. At the same time, the ligustrazine nitrone derivative TBN can increase the movement speed, swing speed, step frequency and stride of D-galactose-induced sarcopenic mice, reduce the time it takes for mice to pass through an equidistant monitoring walkway, and effectively improve the body coordination ability of the sarcopenic mice. The ligustrazine nitrone derivative of the present invention can effectively improve the muscle mass, muscle strength and body function of sarcopenia model animals and has the potential to be developed into a drug for preventing or treating sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows the effects of different doses of TBN on the body weight of D-galactose-induced sarcopenia mice, with the abscissa representing time (weeks) and the ordinate representing body weight (g);

[0024] Figure 2 shows the effects of different doses of TBN on food intake in D-galactose-induced sarcopenia mice, with the horizontal axis representing time (weeks) and the vertical axis representing food intake (g);

[0025] FIG3 is a graph showing the effect of different doses of TBN on the ratio of gastrocnemius muscle wet weight to body weight in D-galactose-induced sarcopenia mice, with the abscissa representing the group and the ordinate representing the ratio of gastrocnemius muscle wet weight to body weight (mg / g);

[0026] Figure 4 shows the effect of TBN on the climbing time of mice with D-galactose-induced sarcopenia in a pole climbing test, with the horizontal axis representing the group and the vertical axis representing the time (s) to climb from the top of the pole to the bottom platform;

[0027] Figure 5 shows the effect of TBN on the limb grip strength of D-galactose-induced sarcopenia mice, with the horizontal axis representing the group and the vertical axis representing the grip strength value (Strength, g);

[0028] FIG6 is a graph showing the effect of TBN on the falling time in the rotarod test in D-galactose-induced sarcopenia mice, with the abscissa representing the group and the ordinate representing the time (s);

[0029] FIG7 is a representative graph of the gait of each group of mice in the TBN-induced sarcopenia mouse gait experiment;

[0030] FIG8 is a graph showing the effect of TBN on the stride length of D-galactose-induced sarcopenia mice, where the abscissa represents the groups of the mice's different limbs and the ordinate represents the stride length (cm);

[0031] FIG9 is a graph showing the effect of TBN on the cadence of D-galactose-induced sarcopenia mice, with the abscissa representing the group and the ordinate representing the cadence (Cadence);

[0032] FIG10 is a graph showing the effect of TBN on the percentage of diagonal support time in D-galactose-induced sarcopenia mice, with the horizontal axis representing the group and the vertical axis representing the percentage of diagonal support time (Time of diagonal support (%));

[0033] FIG11 is a graph showing the effect of TBN on the gait duration of D-galactose-induced sarcopenia mice, with the abscissa representing the group and the ordinate representing the run duration (s);

[0034] FIG12 is a graph showing the effect of TBN on the stand time of D-galactose-induced sarcopenia mice, with the horizontal axis representing grouping and the vertical axis representing stand time (s);

[0035] FIG13 is a graph showing the effect of TBN on the average walking speed of D-galactose-induced sarcopenia mice, with the horizontal axis representing the group and the vertical axis representing the average running speed (cm / s);

[0036] FIG14 is a graph showing the effect of TBN on the swing speed of D-galactose-induced sarcopenia mice, with the abscissa representing the group and the ordinate representing the swing speed (cm / s);

[0037] FIG15 is a representative image of TBN positive staining of HE, Masson and α-SMA in D-galactose-induced sarcopenia mice;

[0038] FIG16 is a diagram showing the statistical results of muscle fibrosis area in Masson staining, wherein the abscissa represents the grouping and the ordinate represents the percentage of muscle fibrosis area (Area of ​​fibrosis, %);

[0039] FIG17 is a statistical diagram of α-SMA positive area, wherein the abscissa represents the grouping and the ordinate represents the percentage of α-SMA positive area (Area of ​​α-SMA, %).

[0040] In the figures of the present specification, ***P<0.001, **P<0.01, *P<0.05 vs. model group. DETAILED DESCRIPTION

[0041] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0042] Example 1

[0043] 1. Preparation of sarcopenia model

[0044] In this example, the sarcopenia model was induced using D-galactose. The experimental animals were intraperitoneally injected with 200 mg / kg of D-galactose at 6 pm every day for 8 weeks, and the control group animals were intraperitoneally injected with an equal amount of normal saline at 6 pm every day.

[0045] 2. Grouping and Dosage Method

[0046] Dasatinib and quercetin were administered together as a positive control. The dose of dasatinib was 5 mg / kg, the dose of quercetin was 50 mg / kg, and the dose of TBN was 10 mg / kg, 30 mg / kg, and 60 mg / kg. The specific groups and doses are shown in Table 1.

[0047] Table 1 Dosage design of this experiment

[0048] Gavage administration. Animals in each group were gavaged starting on the 15th day after D-galactose-induced modeling. The control group, model group, TBN low-dose group, TBN medium-dose group, and TBN high-dose group were gavaged once a day at 9:00 am. The dasatinib + quercetin group was gavaged for 3 consecutive days every two weeks, and each group was treated for 6 consecutive weeks.

[0049] 3. Effect evaluation

[0050] The improvement of sarcopenia motor function by TBN treatment was evaluated through a series of motor behavior tests such as pole climbing, rod rotating, grip strength and gait experiments. The effect of TBN on muscle fibrosis in sarcopenia mice was evaluated by HE staining, Masson staining and α-SMA.

[0051] Graphpad Prism 9.0 software was used for statistical analysis. Data were expressed as Mean ± SEM. One-way ANOVA or Two-way ANOVA was used for comparison among multiple groups. A p value of < 0.05 was considered to indicate a statistically significant difference.

[0052] 3.1 Effects of body weight and gastrocnemius muscle wet weight ratio

[0053] The changes in the body weight and food intake of mice were recorded weekly from before the experimental model was established until the end of behavioral studies. The body weight and gastrocnemius muscle wet weight of mice were recorded when samples were collected.

[0054] The experimental results showed that there was no significant difference in the body weight (Figure 1) and food intake (Figure 2) of mice in each group, indicating that TBN treatment had no significant effect on the body weight and food intake of mice; while the ratio of gastrocnemius muscle wet weight to body weight in D-galactose-induced sarcopenia mice was significantly reduced compared with the control group, while high-dose TBN treatment could increase the ratio of gastrocnemius muscle wet weight to body weight in mice (Figure 3).

[0055] 3.2 Motor function assessment

[0056] 3.2.1 Pole climbing experiment

[0057] After 6 weeks of medication, the pole climbing test was used to test the limb motor ability and muscle strength of the mice. The climbing pole in this experiment was 60 cm long and a wooden pole with a diameter of about 1 cm. The wooden pole was wrapped with medical tape to increase the friction between the mouse and the pole. During the experiment, the mouse was grabbed by the tail so that the mouse's head was facing down. The timing started after the tail was released. The time was based on the forelimbs touching the ground. The time it took for the mouse to climb to the bottom of the pole without external force was recorded. The test time was 15s as the maximum value. If it exceeded 15s, it was counted as 15s. Pole climbing training was carried out 6 weeks after medication. After three consecutive trainings, the mice that did not meet the standards were eliminated. The pole climbing test was carried out on the second day of training. Each mouse was tested three times to obtain the average value.

[0058] 3.2.2 Rotating rod fatigue test

[0059] After 6 weeks of administration, the rotarod test was used to evaluate the motor coordination of mice in a rotating state. A plastic log (6.0 cm in diameter) with a raised shape was fixed at a height of 30 cm. The time of the fall was automatically recorded by the instrument. Before the test, the mice were trained at a speed of 10 rpm / s for 3 days for 3 minutes. After the training, each mouse was tested 3 times on an accelerated rotation (40 rpm per minute). When the mouse could not stay on the rod for 3 minutes, the average falling time was recorded. The average of the 3 falling times of the mouse was the time the mouse stayed on the rotarod.

[0060] 3.2.3 Grip strength test

[0061] After six weeks of dosing, a grip strength test was performed to directly assess the strength of the mouse's limb muscles. The mouse was placed in the center of the grip of the grip strength tester and gently pulled on its tail to encourage it to grasp the grip. When the mouse firmly grasped the grip, the grip was immediately pulled back until the paw released, indicating the mouse's maximum grip strength. The experiment was repeated three times, and the average of the three results was used as the evaluation value.

[0062] The results are shown in Figures 4 to 6. Compared with the control group, the D-galactose-induced sarcopenia mice took longer to climb the pole (Figure 4), had decreased grip strength (Figure 5), and had shorter rotarod falling time (Figure 6), indicating that D-galactose-induced motor function in mice was impaired, and the low, medium, and high doses of TBN could significantly improve the motor function induced by D-galactose.

[0063] 3.2.4 Gait experiment

[0064] After six weeks of dosing, gait analysis was used to assess gait performance and identify abnormal movements in mice. Gait data were collected and analyzed using the CatWalk XT system (Noldus). The system consists of a 1.3-meter-long black corridor and a 70-cm-thick glass panel. A high-speed camera was used beneath the mouse walkway to capture the outlines of mouse paw prints. The walkway was set to a 10 × 20 cm area according to the manufacturer's recommendations. Three days prior to testing, each mouse was allowed to freely walk on the glass panel at least five times daily to acclimate to the gait environment. During testing, the minimum and maximum run times were set to 1 and 10 seconds, respectively, and acquisition was repeated three times per mouse. The same detection settings were used for all groups of mice (camera gain: 20.0, green intensity threshold: 0.10, red ceiling light: 17.7, green channel light: 16.5). For analysis, after gait parameters were automatically generated, each footprint was manually inspected and labeled for the LF (left front), LH (left hind), RF (right front), and RH (right hind) paws. Gait parameters such as movement speed, swing speed, stance and swing time, step length, cadence and leaning support time were collected.

[0065] The gait of each group of mice in the TBN treatment experiment on D-galactose-induced sarcopenic mice is shown in Figure 7 , stride length (cm) statistics are shown in Figure 8 , cadence statistics are shown in Figure 9 , diagonal support time (%) statistics are shown in Figure 10 , run duration (s) statistics are shown in Figure 11 , stand time (s) statistics are shown in Figure 12 , run average speed (cm / s) statistics are shown in Figure 13 , and swing speed (cm / s) statistics are shown in Figure 14 . The results showed that TBN treatment increased the run average speed, swing speed, stride length, and cadence of D-galactose-induced sarcopenic mice, and reduced the run duration and stand time of mice passing through an equidistant monitoring walkway, indicating that TBN can improve the motor coordination ability of sarcopenic mice.

[0066] 3.3 Assessment of muscle fibrosis

[0067] 3.3.1 HE staining

[0068] Mice were deeply anesthetized with 4% chloral hydrate and perfused with normal saline before dissection. The right gastrocnemius muscle was incubated in fresh 4% paraformaldehyde at 4°C overnight, dehydrated using a gradient of alcohol (75% ethanol for 2 hours; 85% ethanol for 1 hour; 95% ethanol for 1 hour; 95% ethanol for 1 hour; 100% ethanol for 0.5 hours; and finally 100% ethanol) and embedded in paraffin. Gastrocnemius muscle tissue was prepared by preparing 5 μm paraffin sections for histological analysis.

[0069] Paraffin sections were first deparaffinized, immersed in xylene, and then rehydrated in graded alcohols. Sections were stained with hematoxylin for 10 minutes, rinsed with tap water for 10 minutes, and stained with eosin for 1 minute. After the sections were thoroughly rinsed in tap water, they were made transparent with ethanol and xylene and finally coverslipped. The stained sections were examined under a microscope and images were captured using a Leica Aperio GT 450 scanner (Leica, Germany).

[0070] 3.3.2 Masson staining

[0071] Paraffin sections were first deparaffinized, immersed in xylene, and then rehydrated in graded alcohols. Dewaxed and rehydrated sections were then stained with Bouin's solution overnight at room temperature and rinsed with water until the yellow color faded. Sections were then drip-stained with lapis lazuli blue for 2 minutes and rinsed with water. Sections were then drip-stained with Mayer's hematoxylin for 2 minutes and rinsed with water. Sections were then drip-stained with an acidic ethanol solution for 10 seconds and rinsed with running water for 10 minutes. Sections were then stained with Ponceau fuchsin for 10 minutes and rinsed with water. Sections were treated with phosphomolybdic acid solution for approximately 10 minutes. Sections were then drip-stained with aniline blue for 5 minutes. Sections were then subjected to weak acid differentiation for 2 minutes. Sections were then dehydrated with graded ethanol, transparentized with xylene, and mounted. Images were captured using a Leica Aperio GT 450 scanner (Leica, Germany).

[0072] α-SMA immunohistochemical staining

[0073] Paraffin sections were first deparaffinized, immersed in xylene, and then rehydrated in graded alcohols. Dewaxed and rehydrated muscle tissue sections were washed (three times with PBS) and subjected to antigen retrieval using 1× antigen retrieval solution and three washes in PBS. Endogenous peroxidases were removed by incubation in hydrogen peroxide for 10 minutes and three washes in PBS. Sections were incubated with 3% BSA (containing 0.3% Triyon-100) at room temperature for 1 hour. Sections were incubated with α-SMA (1:500) antibody at 4°C overnight and four washes in PBS. Sections were incubated with secondary antibody working solution for 1 hour at room temperature and four washes in PBS. Sections were incubated with tertiary antibody working solution for 1 hour and four washes in PBS. Sections were stained with DAB for 2 minutes and washed in PBS. Sections were dehydrated in xylene, transparentized, and mounted. Images were captured using a Leica Aperio GT 450 scanner (Leica, Germany).

[0074] Results: Representative images of HE staining, Masson staining and α-SMA immunohistochemical staining of the gastrocnemius muscles of mice in different groups are shown in Figure 15. In Masson staining and α-SMA immunohistochemical staining, it can be seen that the D-galactose model group has more blue collagen fibers, darker α-SMA staining and a larger positive area, while TBN treatment can reduce the fibrosis area. The statistical analysis of the percentage of collagen fiber area (the percentage of positive area to the total area of ​​the statistical area) and the percentage of α-SMA positive area (the percentage of positive area to the total area of ​​the statistical area) of each group of mice are shown in Figures 16 and 17. The results suggest that TBN can improve the gastrocnemius muscle fibrosis of D-galactose-induced sarcopenia model mice.

[0075] Referring to the above experiments, it was found that TN-2 can also effectively improve the muscle mass, muscle strength and muscle function of sarcopenia model animals, and has the potential to be developed into a drug for the prevention or treatment of sarcopenia.

Claims

1. Use of a ligustrazine nitrone derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating sarcopenia; the ligustrazine nitrone derivative has a structure shown in the following general formula (I): in, R1 is hydrogen, methyl or R2 and R3 are the same or different and are independently selected from hydrogen or C1-C6 alkyl; R4 is sec-butyl, isobutyl, tert-butyl, cyclopentyl or cyclohexyl, and R5 is sec-butyl, isobutyl, tert-butyl, cyclopentyl or cyclohexyl.

2. The use according to claim 1, characterized in that: R2, R3 are the same or different and are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or n-pentyl.

3. The use according to claim 1, characterized in that The ligustrazine nitrone derivative is TBN or TN-2:

4. The use according to any one of claims 1 to 3, characterized in that: The sarcopenia is primary sarcopenia or secondary sarcopenia.

5. The use according to any one of claims 1 to 3, characterized in that: The drug is used to reduce the loss of muscle mass caused by sarcopenia, and to maintain or increase muscle mass.

6. The use according to any one of claims 1 to 3, characterized in that: The drug is used to improve muscle strength in sarcopenia.

7. The use according to any one of claims 1 to 3, characterized in that: The drug is used to improve the physical function of sarcopenia patients.

8. The use according to any one of claims 1 to 3, characterized in that: The medicament is used for treating or preventing muscle fibrosis in sarcopenia.

9. The use according to any one of claims 1 to 3, characterized in that: Comprising administration of 100-5000 mg of ligustrazine nitrone derivatives per day.

10. The use according to any one of claims 1 to 3, characterized in that: The pharmaceutically acceptable salt is a salt formed by a ligustrazine nitrone derivative and an acid, wherein the acid is selected from hydrochloric acid, hydrobromic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, phosphoric acid, acetic acid, propionic acid, benzoic acid, hexanoic acid, hydroiodic acid, nitric acid, sulfuric acid, salicylic acid, oxalic acid, malonic acid, tartaric acid, stearic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, cinnamic acid, 2-naphthalenesulfonic acid, succinic acid, D-gluconic acid, dodecyl sulfuric acid, pyrosulfuric acid, pyruvic acid, cyclopentanepropionic acid, citric acid, lactic acid or aspartic acid.

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