Application of ginsenoside Rb1 in preparation of medicine for treating amyotrophic lateral sclerosis

Abstract

The invention discloses application of ginsenoside Rb1 in preparation of a medicine for treating amyotrophic lateral sclerosis and application of ginsenoside Rb1 in treatment of neurodegenerative diseases related to dyskinesia. A large number of experiments show that the ginsenoside Rb1 can improve the dyskinesia state of hSOD1G93A model mice in behavioral experiments such as Rotard bar rotating experiments, gait monitoring experiments and cage experiments, and the life cycle of SOD1 model caenorhabditis elegans is effectively prolonged.

Description

Technical Field

[0001] The present invention relates to the field of pharmacological therapy, and more particularly to the use of ginsenoside Rb1 in the preparation of a drug for treating amyotrophic lateral sclerosis. More particularly, the present invention relates to the effect of ginsenoside Rb1 on improving SOD1 G93A The invention discloses a model mouse with motor dysfunction, and is used for preparing a drug for treating amyotrophic lateral sclerosis caused by superoxide dismutase 1 (SOD1) gene mutation. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that mainly affects the spinal cord, cerebral cortex and brainstem motor neurons. ALS usually occurs in middle-aged or elderly people or later. After the onset of the disease, patients will show progressive muscle atrophy and weakness, and eventually die of respiratory failure. Most patients only survive for 2 to 4 years after the onset of the disease.

[0003] Multiple genes are associated with ALS, and mutations in the SOD1 gene are the second most common cause of familial ALS, accounting for approximately 10%-20% of familial ALS cases and 1%-2% of all ALS cases. In SOD1-ALS patients, mutations in the SOD1 gene cause the patient's body to produce a toxic, misfolded form of the SOD1 protein. This toxic protein causes motor neuron degeneration, leading to progressive muscle weakness, loss of function, and ultimately death. In 1993, a study found that mutations in the SOD1 gene were the causative gene for ALS. Based on this, the first ALS model mouse, hSOD1, was developed in 1994. G93A Model mice were developed and have been widely used in ALS research.

[0004] ALS is a rare disease, but due to its extremely low cure rate, it has brought great pain to ALS patients and their families. Currently, there are only two drugs widely used in the clinic to treat ALS. Among them, riluzole was approved for marketing as early as 1995, but it was only approved by the FDA for the treatment of ALS in recent years. Another drug, edaravone, was approved by the FDA for the treatment of ALS in 2017. For ALS patients related to SOD1 gene mutations, the gene therapy drug Tofersen, which has been approved for marketing in recent years, can bind to the mRNA encoding SOD1, causing it to be degraded by the ribonuclease RNase-H, thereby reducing the production of mutant SOD1 protein and playing a role in treating ALS. More than a year after Tofersen was approved in the United States, on May 30, 2024, Biogen announced that Tofersen has been approved by the European Union for the treatment of adult amyotrophic lateral sclerosis (ALS, also known as "Lou Gestational Frost Syndrome") with superoxide dismutase 1 (SOD1) gene mutations. The drug was approved through the accelerated approval pathway and is the first therapy approved by the European Union for the genetic cause of ALS.

[0005] Further exploring the pathogenic mechanism of ALS, finding new targets for treating the disease, and developing drugs that are effective in treating ALS and accessible to the public have important social value and practical significance. Summary of the invention

[0006] The present invention provides a new medical application of ginsenoside Rb1, namely, application of ginsenoside Rb1 in preparing a medicine for treating amyotrophic lateral sclerosis.

[0007] In some embodiments, use of ginsenoside Rb1 as the sole active ingredient in the preparation of a medicament for treating amyotrophic lateral sclerosis.

[0008] In some embodiments, the amyotrophic lateral sclerosis is amyotrophic lateral sclerosis with a superoxide dismutase 1 (SOD1) gene mutation (SOD1-ALS). Preferably, the amyotrophic lateral sclerosis is adult amyotrophic lateral sclerosis with a superoxide dismutase 1 (SOD1) gene mutation. Preferably, the mutation is a G93A mutation or a G85R mutation.

[0009] In some embodiments, the ginsenoside Rb1 can improve movement dysfunction.

[0010] In some embodiments, the ginsenoside Rb1 can improve hSOD1 G93A Model of motor dysfunction in mice.

[0011] In some embodiments, the medicament includes a pharmaceutically acceptable excipient.

[0012] In some embodiments, the medicament is a tablet, capsule or injection.

[0013] Ginsenoside Rb1, its structural formula is:

[0014]

[0015] Ginsenoside Rb1 is currently used to treat amyotrophic lateral sclerosis, especially amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutation, and improve hSOD1 G93A The effects of motor dysfunction in model mice have not been reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Ginsenoside Rb1 can significantly improve hSOD1 G93A The model mice showed motor dysfunction in the Rotard rod test. *** represents p < 0.001, compared with the blank group; # represents p<0.05, ### Represents p < 0.001, compared with the model group. One-way ANOVA.

[0017] Figure 2 Ginsenoside Rb1 can significantly improve hSOD1 G93 A model mice exhibit abnormal gait in the gait monitoring experiment. * represents p<0.05, ** represents p<0.01, *** represents p < 0.001, compared with the blank group; ## represents p<0.01, ### Represents p < 0.001, compared with the model group. One-way ANOVA.

[0018] Figure 3 Ginsenoside Rb1 can significantly improve hSOD1 G93A The model mice showed decreased grip strength in their limbs in the hanging cage experiment. *** represents P < 0.001, compared with the blank group; # Represents p < 0.05, compared with the model group. One-way ANOVA.

[0019] Figure 4 Ginsenoside Rb1 can significantly prolong the survival period of the SOD1 model Caenorhabditis elegans. * represents p < 0.05, compared with the blank group; # Represents p < 0.05, compared with the model group. Two-way ANOVA. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific examples, but these examples should not be construed as limiting the present invention.

[0021] Example: Ginsenoside Rb1 improves hSOD1 G93A The model mice showed a state of motor dysfunction in behavioral experiments such as the Rotard rod test, gait monitoring test and hanging cage test, and effectively delayed the life cycle of the SOD1 model Caenorhabditis elegans.

[0022] The present invention is in hSOD1 G93A The effect of ginsenoside Rb1 on the improvement of motor dysfunction in mice was tested in model mice. The experiment showed that ginsenoside Rb1 significantly improved hSOD1 G93A The model mice showed motor dysfunction in behavioral experiments such as the Rotard rod test, gait monitoring test and hanging cage test.

[0023] 1. Experimental Principle

[0024] 1) Rotard rod test: hSOD1 G93A The model mice will have motor dysfunction when they become ill, so the animal's forelimb and hindlimb motor coordination and balance function can be assessed by detecting the length of time the mice stay on the rotating rod.

[0025] 2) Gait monitoring experiment: hSOD1 G93A The model mice showed unstable gait and resting tremor at the beginning of the disease, which gradually developed into hind limb atrophy. In the late stage of the disease, it developed into complete paralysis of both hind limbs and a significantly shortened stride length. Gait analysis was used to evaluate the motor coordination function of the mice's forelimbs and hindlimbs.

[0026] 3) Cage experiment: hSOD1 G93A After the onset of the disease, the model mice showed atrophy of their hind limbs and decreased grip strength of their limbs. The hanging cage experiment was used to test the grip strength of the mice's limbs.

[0027] 4) C. elegans survival cycle detection: The time from onset to death of SOD1 model C. elegans is relatively short. Whether the survival cycle can be effectively extended is an important indicator for evaluating the anti-ALS effect of a compound.

[0028] 2. Experimental Materials and Methods

[0029] 1) Animal grouping: Experimental mice B6SJL-BTg (hSOD1 G93A )1Gur / J (002726) was purchased from Jackson Laboratory, USA. hSOD1 was obtained by gene identification G93AA total of 36 mice were randomly divided into 5 groups, including wild-type mice of the same litter as negative control. They were: ① wild-type solvent group (WT+Vechile group, n=12), ② wild-type drug group (WT+Rb115 mg / kg / day group, n=12), ③ model solvent group (Tg+Vechile group, n=12), ④ model low-dose drug group (Tg+Rb17.5 mg / kg / day group, n=12) and model high-dose drug group (Tg+Rb115 mg / kg / day group, n=12). All mice were drugged from 56 days of age (8 weeks old) and killed at 120 days of age. Blood, muscle and spinal cord tissues were collected from the eyeball for pathological, biochemical and immunological analysis. Experimental Caenorhabditis elegans SOD1-WT and SOD1-G85R were provided by the Caenorhabditis Genetics Center (CGC) in the United States. With C. elegans SOD1-WT as the control group and C. elegans SOD1-G85R as the model group, they were divided into 5 groups as follows: ① wild-type solvent group (SOD1-WT+Vehicle group, n=50), ② model solvent group (SOD1-G85R+Vehicle group, n=50), ③ model low-dose administration group (SOD1-G85R+Rb125μM / day group, n=50) and ④ model high-dose administration group (SOD1-G85R+Rb150μM / day group, n=50). When the C. elegans in the culture dish is observed to have eggs under a microscope, use 1ml M9 buffer to rinse the C. elegans in the culture dish and transfer it to a 1.5ml EP tube. Remove the supernatant after natural sedimentation. After washing twice with M9, use ddH2O to wash, remove the supernatant, and start lysis. The ddH2O is three times the volume of the nematodes, 100% hypochlorous acid is one time the volume of the nematodes, and sodium hydroxide is half the volume of the nematodes. Vortex for 5-8 minutes after adding, and the whole step should not exceed ten minutes. Too long will damage the eggs. The liquid will turn yellow after lysis. Observe the floating eggs under a microscope, centrifuge at 6000 rpm for 1-2 minutes, and wash two or three times with M9 buffer after centrifugation. The lysed worm bodies were blown evenly with 2 ml of M9 buffer and transferred to a small dish. After culturing in an incubator for 24 hours, the nematodes were F1, and the M9 buffer containing nematodes in the small dish was transferred to a 1.5 ml EP tube. After centrifugation, most of the supernatant was removed, and the remaining nematodes were transferred to NGM culture medium. After 60 hours, all nematodes were synchronized adults of the F4 period, i.e., the L4 stage. The synchronized L4 stage Caenorhabditis elegans was placed on the NGM plate, and the second day was defined as the first day. Different concentrations of ginsenoside Rb1 were given every day, and Caenorhabditis elegans that did not respond to external mechanical stimulation were judged to be dead. There was no record of Caenorhabditis elegans crawling out of the NGM plate.All C. elegans were transferred to new NGM plates every other day until all model C. elegans died and their survival cycles were analyzed.

[0030] 2) Rotarod test: Starting from 60 days of age, hSOD1 G93A The motor function of model mice was tested. The mice received a week of training to familiarize themselves with the Rotarod instrument. Starting from 70 days of age, the test was conducted every two days. At the beginning of the experiment, the mice were placed individually on the rotating cylinder of the rotarod instrument and rotated at a constant speed of 12 revolutions per minute. The motor coordination and balance of each mouse were evaluated by measuring the total movement time of the mouse on the rotating rod. Each animal was tested three times, and the longest stay time before falling was recorded, and the maximum stay time was set to 180 seconds.

[0031] 3) Gait monitoring experiment: In order to obtain footprints, hSOD1 G93A The front and hind feet of the model mice were painted with red and green non-toxic paints, respectively. The mice walked along a 50 cm long and 10 cm wide track and the gait of the mice was recorded. All mice were tested once a week, running three times each time. The mouse footprints were analyzed, and the average of the three results was used in the analysis of the step length of the mice.

[0032] 4) Cage experiment: Test once a week starting from the 8th week. During the experiment, a soft cushion was laid on the ground to protect the mice. Then the mice were placed on the cage cover. After the mice grasped the cage cover, the cage cover was quickly turned over and the timing was started at the same time. The longest time the mice grasped the cage cover was recorded. The maximum time was set to 90 seconds. If it was less than 90 seconds, the specific time was recorded. Each experiment was repeated three times and the best result was recorded.

[0033] 5) Life cycle detection: The death date of C. elegans is defined as the day when C. elegans does not respond to external mechanical stimulation and is recorded as the death date of C. elegans.

[0034] 3. Experimental results

[0035] The results are as follows Figure 1 As shown in Figure 2, treatment with ginsenoside Rb1 significantly increased hSOD1 G93A The model mice stayed on the rotating rod for a longer time (p < 0.05, p < 0.001), indicating that ginsenoside Rb1 has the effect of improving the motor coordination and balance function of ALS model mice; Figure 2 As shown in Figure 2, treatment with ginsenoside Rb1 significantly increased hSOD1 G93A The stride length of model mice indicated that ginsenoside Rb1 could improve the abnormal gait of ALS model mice (p<0.01, p<0.001); Figure 3As shown in Figure 2, treatment with ginsenoside Rb1 could significantly prolong the hSOD1 G93A The persistence time of model mice in the hanging cage experiment (p < 0.05) indicated that ginsenoside Rb1 has the function of enhancing the muscle strength of ALS model mice; Figure 4 As shown, treatment with ginsenoside Rb1 can prolong the time that SOD1 Caenorhabditis elegans does not respond to external mechanical stimulation (p < 0.05), indicating that ginsenoside Rb1 has the effect of prolonging the survival period of ALS model Caenorhabditis elegans.

[0036] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. Use of ginsenoside Rb1 as the sole active ingredient in the preparation of a drug for treating amyotrophic lateral sclerosis.

2. The use according to claim 1, wherein The amyotrophic lateral sclerosis is amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutation.

3. The use according to claim 2, wherein the mutation is a G93A mutation or a G85R mutation.

4. The use according to any one of claims 1 to 3, wherein the medicament comprises a pharmaceutically acceptable excipient.

5. The use according to any one of claims 1 to 3, wherein the medicine is a tablet, a capsule or an injection.

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