Traditional Chinese medicine treatment for amyotrophic lateral sclerosis (ALS).

Abstract

Traditional Chinese medicine treats amyotrophic lateral sclerosis (ALS). This medicine is formulated by combining Ginseng radix et rhizoma with Cistanches herba.

Description

A Chinese medicine composition for treating amyotrophic lateral sclerosis and its application

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on March 2, 2023, with application number 202310190284.4 and invention name “A Chinese medicine composition for treating amyotrophic lateral sclerosis and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition for treating amyotrophic lateral sclerosis and application thereof. Background Art

[0003] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology that primarily affects motor neurons in the cerebral cortex, brainstem, and spinal cord. Its primary clinical manifestations are progressive skeletal muscle weakness, atrophy, fasciculations, bulbar palsy, and pyramidal tract signs. The average survival period is 3 to 5 years. Approximately 5 to 10% of cases are familial (FALS), while 90 to 95% are sporadic (SALS). The global incidence of ALS is approximately 1.59 per 100,000 people, with a prevalence of 4.42 per 100,000. In 2020, analysis of medical insurance data revealed that the total number of ALS patients in my country was approximately 40,000, making it a rare disease. The disease has an insidious onset, and the risk of onset increases with age. It is progressive and fatal, rapidly worsening after symptoms appear. The World Health Organization currently lists ALS as one of the five most incurable diseases, along with cancer, AIDS, leukemia, and rheumatoid arthritis. Clinical diagnosis is difficult, with high rates of misdiagnosis and missed diagnosis, and treatment is expensive. A survey of the economic burden of ALS patients in southwest China revealed that the average annual cost per person is 85,400 RMB, far exceeding the average annual income of Chinese residents (35,128 RMB in 2021). As the disease progresses, ALS patients lose the ability to care for themselves, placing a heavy burden on their families and society. There is an urgent need to find effective treatments and medications to slow the disease's progression and improve patients' quality of life.

[0004] The primary pathological manifestations of ALS are partial or complete loss of giant pyramidal cells in the bilateral precentral gyri of the cerebral cortex, degeneration of brainstem motor nuclei, axonal degeneration of the central nervous system, and neurogenic muscle atrophy. Physiologically, neurotransmitters synthesized within motor neuron cell bodies are transported along axons to the distal terminals and transmit excitation to skeletal muscle via the neuromuscular junction, thereby causing skeletal muscle excitation and contraction. When factors such as gene mutations, glutamate excitotoxicity, oxidative stress, and mitochondrial dysfunction lead to motor neuron damage and neuromuscular junction dysfunction, neurotransmitter transmission is blocked, muscle damage and contraction are inhibited, leading to the development of ALS. The pathogenesis of ALS involves various stages from presynaptic neuronal lesions to neuromuscular junction damage, and these stages are interconnected and mutually influential, ultimately resulting in a pathology primarily characterized by damage to the motor nervous system.

[0005] Rare diseases like ALS require early diagnosis and treatment to maximize patient survival. The European Medicines Agency's "Clinical Guidelines for the Treatment of ALS" proposes that the primary treatment goals for ALS are to improve survival, slow disease progression, and improve ALS symptoms (muscle strength and related function). In 2019, the National Health Commission issued the "Guidelines for the Diagnosis and Treatment of Rare Diseases," recommending riluzole and edaravone as treatments for ALS. Riluzole is a glutamate inhibitor with common adverse reactions including fatigue and nausea. Some patients may experience elevated liver transaminases. It can prolong survival by an average of approximately three months, but short-term use is not effective, requiring treatment for more than six months to improve prognosis. Edaravone is a free radical scavenger primarily indicated for early-stage patients within two years of onset and with a forced vital capacity (FVC) greater than 80% of predicted. Its clinical benefits are limited, and clinical studies have shown that long-term (8.9 to 13.9 months) edaravone infusions do not improve disease progression. A new combination preparation of sodium phenylbutyrate and taurine diol was approved in 2022. Its clinical efficacy needs to be further verified. The instructions record common adverse reactions as diarrhea, abdominal pain, nausea and upper respiratory tract infection. A total of 247 records were retrieved from the Yaodu database using the search term "amyotrophic lateral sclerosis", of which only Masitinib, Tofersen and Arimoclomol were drugs that applied for marketing approval, accounting for 1%. Drugs in clinical phase III accounted for 6%, phase II accounted for 13.4%, preclinical studies accounted for 31.6%, non-progression status accounted for 9.7%, and termination status accounted for 11.3%. Masitinib has been shown to have the effect of delaying disease progression, but the safety of this product still needs further verification. Tofersen did not reach the primary endpoint, and only a trend of reducing disease progression was observed.

[0006] ALS, based on clinical symptoms such as decreased muscle strength and atrophy, falls under the category of "weakness" in Traditional Chinese Medicine (TCM). Traditional Chinese medicine defines "weakness" in two ways: first, referring to the disease itself, also known as "weakness and paralysis." The "Suwen: Discussion on Weakness" categorizes it as "five kinds of weakness": skin weakness, pulse weakness, tendon weakness, muscle weakness, and bone weakness. Second, it refers to symptoms. "Suwen: Discussion on Yin and Yang" states that "the onset of the three yin and three yang syndromes is characterized by hemiplegia and weakness, with the limbs becoming incapable of movement." Liu Hejian, in "Suwen: Mysterious Origin of Disease Patterns," states: "Weakness refers to the weakness of the hands and feet, the inability to move." This describes "weakness" as a condition characterized by the weakening of the tendons and veins in the limbs. Regarding the understanding of the pathogenesis of this disease, doctors of all generations mostly took the "Inner Canon of Medicine" that the five internal organs cause paralysis as the general outline, and formed the understanding of the pathogenesis such as lung heat damaging body fluid, damp heat infiltration, spleen and stomach weakness, liver and kidney deficiency, etc. Among them, the scattered discussions on treating paralysis from the extraordinary meridians provide clinical treatment experience for reference for the treatment of such difficult diseases.

[0007] During the Warring States Period, the "Inner Canon of Medicine" and "Difficult Classic" proposed that impotence can be divided into deficiency and excess, and into the five internal organs, established the treatment method of "treating impotence by treating only Yangming", and proposed that impotence is related to Chong, Du and Dai meridians, which became the beginning of treating impotence from the extraordinary meridians; during the Tang and Song Dynasties, impotence was treated from the perspective of internal organ deficiency, and the idea of ​​using tonic drugs as the main treatment was formed; during the Jin and Yuan Dynasties, emphasis was placed on the important pathogenesis of spleen, stomach, liver and kidney in impotence, and the syndrome differentiation and treatment prescriptions were enriched; Ming and Qing doctors treated impotence from the perspective of deficiency of vital energy, extraordinary meridians and qi, which promoted the further development of impotence research in traditional Chinese medicine. Doctors of all dynasties have accumulated rich experience in treating impotence from the five internal organs. They have analyzed the internal and external causes, and the symptoms of deficiency and excess, and mostly focused on the damage of lung heat and body fluid, infiltration of damp heat, weakness of the spleen and stomach, and deficiency of the liver and kidney, which lead to lack of nourishment of muscles and tendons, sluggish tendons and veins, and flaccid limbs. They formed the treatment methods and representative prescriptions of "treating impotence by only taking Yangming", clearing the lungs and moistening dryness, clearing away damp heat, nourishing the spleen and stomach, and nourishing the liver and kidneys, especially those that replenish vital energy and nourish the liver and kidneys.

[0008] However, looking through ancient records, doctors of all generations only discussed treating paralysis through the extraordinary meridians and collaterals sporadically in the Neijing (Inner Canon of Medicine) and during the Ming and Qing dynasties, but this approach was not further applied or developed. As a result, for a long period of history, the impact of the extraordinary meridians and collaterals on the pathogenesis of this disease and their role in treatment did not receive sufficient attention, nor was systematic research conducted. This was not until the famous Qing Dynasty physicians Ye Tianshi and Xu Yutai proposed that "muscle paralysis and bone paralysis are all related to the extraordinary meridians and collaterals" (Essence of Medical Talks by Famous Qing Dynasty Physicians, Xu Yutai). However, the explanation was vague and the rationale was not thoroughly explored. In fact, the eight extraordinary meridians store and overflow Qi and blood in the twelve meridians, and have the function of coordinating the rise and fall of Yin and Yang Qi and blood throughout the body. Starting from regulating the extraordinary meridians and unblocking the collaterals is conducive to explaining the TCM pathogenesis of this disease and is expected to explore new treatment strategies. This is of great significance in improving the quality of life and clinical symptoms of this type of patients.

[0009] In summary, ALS, as a rare disease that seriously threatens the lives of patients, faces severe problems such as lack of clinical treatment drugs, uncertain efficacy, obvious side effects, and difficulty in developing new drugs. Therefore, the development of innovative drugs to effectively improve clinical symptoms and improve quality of life is urgent and has become a major issue that needs to be urgently addressed in clinical practice.

[0010] Summary of the Invention

[0011] Academician Wu Yiling proposed the "Treatment of Motor Neuron Disease from the Peripheral Meridians" approach, providing systematic theoretical guidance for a variety of neuromuscular diseases, including ALS. Deficiency of the Extraordinary Meridians and malnutrition of the Eight Meridians can lead to a loss of the warming and nourishing Qi, Blood, Yin, and Yang of the twelve meridians and the five internal organs, resulting in paralysis and weakness, particularly closely related to the Governor Meridian. Furthermore, paralysis is associated with internal organ deficiency, leading to the "Five Internal Organs Syndrome" approach. Starting from the clinical symptoms and signs of ALS, this approach comprehensively analyzes the dysfunction of the Extraordinary Meridians and internal organs, identifying key pathological mechanisms such as deficiency of Yuan Jing, deficiency of lung and stomach fluid, lung heat and leaf scorch, and stomach heat and Yin deficiency. Combining the principles of "treating the three burners separately," the upper burner should be lifted, either by clearing dryness and relieving the lungs, replenishing qi and producing fluid to aid the lungs' ability to disperse and spread, or by replenishing qi and lifting sunken qi to aid the great qi's mediation, or by nourishing the heart's blood to nourish all meridians. The middle burner should be transformed, either by strengthening the spleen and promoting circulation to nourish the source of transformation, or by clearing dampness and heat to unblock the middle burner's stagnation. The lower burner should be filled, warming and regulating the extraordinary yang to invigorate the depleted true essence, and warming and replenishing the true essence to nourish the depleted yin and blood. These insights provide new insights into the treatment of impotence and have important guiding value for ALS. The eight extraordinary meridians hold significant and unique value in the treatment of impotence, especially the Du meridian. It replenishes the spinal cord, transformed from kidney essence, and forms brain marrow in the brain. Its course connects the kidneys and brain, connecting the upper and lower parts, coinciding with the location of ALS lesions. Academician Wu Yiling innovatively proposed the "extraordinary meridians to treat impotence, five internal organs to differentiate symptoms, and triple burner to differentiate symptoms". He treats impotence based on the theory of the eight extraordinary meridians and the governing vessel. This not only highlights the theoretical characteristics of the eight extraordinary meridians in traditional Chinese medicine, but also absorbs the understanding of the disease by modern medicine, integrating Chinese and Western medicine, making the exploration of pathogenesis and treatment more relevant to clinical practice and effectively guiding clinical practice.

[0012] Academician Wu Yiling has been dedicated to the study of the theory of collateral disease and its applications since the 1980s. He categorizes diseases characterized by a long course, recurring attacks, and prolonged illnesses as collateral disease, a category that ALS falls into. Collateral disease theory states that collaterals, as a network system that branches out from the meridians, are gradually subdivided, crisscross, and widely distributed throughout the body, encompassing organs and tissues. They are the network structures that sustain life and maintain a stable internal environment. Based on their functions in circulating qi and blood, they are divided into collaterals of meridians and collaterals of veins, forming two major branches of research: the Qi-collateral theory and the Vein-collateral theory. The Qi-collateral theory proposes that qi in the meridians is meridian qi, and that qi entering the collaterals is collateral qi. Meridian qi diffuses through the Qi-collateral surfaces to the organs, skin, limbs, and bones, fulfilling functions such as collateral regulation, warmth and nourishment, defense, information transmission, and homeostasis. Qi-collateral disorders primarily involve abnormalities in the brain, marrow, and Du channel, endocrine metabolism, and immune function.

[0013] As a unique network structure, the Qi and collaterals are directly connected to the meridians and brain marrow. As stated in the Lingshu: "Evil Qi and Organ Diseases" (Lingshu: The Forms of Diseases in the Internal Organs), "The twelve meridians and three hundred and sixty-five collaterals, their blood and Qi all ascend to the face and flow through the empty orifices." Collaterals also serve as crucial pathways connecting the extraordinary meridians and brain marrow with the muscles, tendons, and limbs. The Qi and collaterals are closely linked to the brain and Du meridians, forming a neural regulatory mechanism with the brain as the apex of Qi movement, the spinal cord as the pathway for Qi movement, and the brain and marrow Qi and collaterals regulating the complex vital movements of the internal organs and externally controlling the limbs.

[0014] The Du channel is closely related to the spinal cord in modern medicine in terms of anatomical location and function. The Du channel runs along the middle of the back and overlaps with the spine. It connects to the brain above and the kidneys below, becoming the "pathway for the rise and fall of essence and energy."

[0015] The brain's ability to regulate motor function relies on the Du channel, which serves as the pathway for the ascending and descending of vital essence and qi. The term "fine collaterals" aligns closely with modern neuroanatomy and histology, which describe spinal nerves branching out from the spinal cord and then gradually extending into the body's interior and exterior. This suggests that the spinal cord and the Du channel share a common course, and the fine collaterals branching out from the spinal cord clearly belong to the Du channel.

[0016] It can be seen that Yuan Qi is generated from the true essence in the kidneys, and enters the brain through the Governor Vessel (marrow). The function of the true Yuan Qi in the brain's Qi and meridians reflects the role of the brain spirit. The brain spirit transmits its regulated divine mechanism along the Governor Vessel Qi to the five internal organs and limbs, thus forming a close connection with the brain and spinal cord nerves that regulate motor function. If Yuan Jing is insufficient, the true Yuan is deficient, the Governor Vessel is not nourished, and the Qi and meridians are not nourished, then paralysis can occur. ALS, as a group of degenerative diseases of the central nervous system, is characterized by the disappearance of pyramidal cells in the motor cortex of the brain, the loss of motor neurons in the anterior horn of the spinal cord and the brainstem, abnormal cellular pathological changes, and degeneration of the corticospinal tract, which in turn leads to muscle atrophy. It can be seen that the main sites of ALS disease are the brain and spinal cord, which are very consistent with the site of brain and spinal cord circulation. The decline of true Yuan, the deficiency of Qi and Yang, and the malnutrition of the brain and marrow will lead to paralysis and weakness of the limbs, muscle atrophy, fatigue and laziness. Therefore, ALS is a Qi and meridian-brain marrow Governor Vessel disease.

[0017] The TCM pathogenesis of ALS, characterized by "insufficient Zhen Yuan and malnutrition of the Du Meridian," was first proposed. Zhen Yuan deficiency is the root cause of the disease: Zhen Yuan Qi, or primordial Qi, is generated from Yuan Jing and circulates through the meridians to the three burners and distributes to the internal organs. Zhen Yuan Qi, the fundamental driving force of human life, stimulates the functional activities of internal organs and tissues through the regulation of the central nervous system. ALS is located in the brain marrow, and innate deficiency of essence directly leads to a deficiency of Yuan Qi and Yuan Yang. Furthermore, the disease often occurs in middle-aged and elderly people, whose essence and energy are depleted, Zhen Yuan is weakened, and organ function declines. Furthermore, improper nourishment, exposure to external pathogens, physical injury, emotional imbalance, dietary restrictions, or excessive sexual activity can further damage Yuan Qi, leading to a deficiency in the brain marrow and dysfunction of its functions, thus emphasizing the root cause of the disease.

[0018] In terms of clinical manifestations, when the True Yuan is depleted, the Yang's active and invigorating functions decline, and the body's active parts become unable to function, manifesting as limb weakness, difficulty walking, fatigue, laziness, and shortness of breath. "Yang transforms into Qi, and Yin forms"; with prolonged Yang deficiency, Yin Qi cannot be generated. Insufficient Yuan Yang and Yuan Qi throughout the body lead to a lack of motivation, a lack of Qi and blood production, and obstructed distribution, unable to nourish the muscles and skin, resulting in muscle weakness and atrophy. With Yang Qi deficiency, the tendons and veins lose their warmth and nourishment, resulting in increased muscle tone and muscle stiffness. Common symptoms of this disease include medullary paralysis and dysphagia, which are caused by insufficient True Yuan, the failure of the clear Yang to rise, the failure of the turbid Yin to descend, the disordered Qi movement, and a blocked pharyngeal opening.

[0019] Malnutrition of the Governor Vessel is the key to the pathogenesis: ALS is located in the brain marrow, and the Governor Vessel of the Extraordinary Meridians runs through the bone marrow and brain. Zhenyuan Qi originates from the kidneys and is controlled by the Governor Vessel. Damage to the Governor Vessel and its branches, resulting in insufficient Zhenyuan Qi, not only weakens its ability to command and regulate the body's Yang Qi, but also severely impacts the areas along its course, causing the spinal cord and brain to lose warmth and nourishment, leading to disease. The Governor Vessel runs through the spine and neck to the brain. Malnutrition of the Governor Vessel can manifest as neck weakness, or even lumbar weakness, and lower back and knee pain. Deficiency of the Extraordinary Meridians and malnutrition of the Governor Vessel weakens its ability to command and regulate the body's Yang Qi, leading to fatigue and laziness. Muscles lack warmth and nourishment, resulting in muscle twitching. Malnutrition of the Governor Vessel, preventing the ascending of Clear Yang, and dysfunction of the upper orifices can cause slurred speech, difficulty swallowing, and coughing when drinking water.

[0020] In summary, the warming, promoting, and nourishing effects of Zhenyuan Qi are fundamental to the normal functioning of the internal organs. The Governor Channel (Du Luo) runs through the spine and connects to the brain, acting as the "sea of ​​yang meridians." It serves as the governor and guides the activities of other meridians and internal organs. The brain follows the rise and fall of the Governor Channel's Qi, connecting to the skin, flesh, tendons, and bones, regulating the motor function of the limbs. If Zhenyuan Qi is deficient, the Governor Channel loses its nourishment, and Yuan Qi fails to reach the muscles, the muscles lose their vital energy and become unable to move. A weakened Governor Channel leads to a deficiency of Extraordinary Yang, weakening the function of Yang Qi throughout the body. The areas along its course are particularly affected, leading to a loss of warmth and nourishment in the spinal cord and brain, which in turn leads to disease. This is highly consistent with the pathology of ALS, characterized by damage to upper and lower motor neurons and bulbar paralysis. Therefore, exploring the etiology and pathogenesis of ALS from the perspective of the Extraordinary and Du Luo channels opens up new research avenues for its treatment.

[0021] Establishing the "Supporting Yuan Qi, Healing Atrophy, and Nourishing the Rong and Regenerating Muscles" treatment approach. In response to the fundamental pathogenesis of ALS, "insufficient Zhen Yuan and malnutrition of the Du Luo," the "Supporting Yuan Qi, Healing Atrophy, and Nourishing the Rong and Regenerating Muscles" treatment approach was developed. Pathologies of the extraordinary meridians are often caused by insufficient or depleted Yuan Qi. Treatment should prioritize supporting Yuan Qi. Supporting Zhen Yuan ensures abundant Yuan Qi, vigorous Yang Qi, unblocked meridians, and nourishment of all internal organs. Therefore, selecting ginseng as a vital Qi tonifier aims to, first, nourish Yuan Yang to replenish the Du Luo; second, replenish the Qi of the five internal organs, allowing the essence to be generated and the body to flourish; and third, invigorate Qi and unclog the blood vessels. Circulating Qi leads to blood circulation, which replenishes Qi and blood, unclogs the blood vessels, replenishes the extraordinary meridians, and replenishes the Qi and blood of the twelve meridians, nourishing the skin and muscles, thus achieving the goal of supporting Yuan Qi and heal atrophy. Therefore, it is advisable to seek yang in yin and seek yin in yang, to strengthen the Yuan and cure impotence, and at the same time use medicines that nourish and replenish the true essence. It can not only nourish the kidney and replenish the essence and blood, but also make the yang generate the yin and replenish the yin in the medicine that nourishes the yin essence, and use it together to strengthen the Yuan Qi and nourish the kidney and fill the essence, so as to achieve the purpose of strengthening the Yuan and curing impotence, nourishing the flesh and regenerating the skin.

[0022] The pharmaceutical composition of the present invention is composed of two medicinal herbs, ginseng and cistanche deserticola. Ginseng tastes sweet and slightly bitter, is neutral in nature, and enters the spleen, lung, and heart meridians. It has the function of greatly replenishing vital energy and nourishing qi and collaterals. The pathogenesis of impotence is due to deficiency of the internal organs and deficiency of the extraordinary meridians and governing channel. Ginseng is the main medicine. First, it can greatly replenish vital energy and nourish primordial yang, and is mainly used to treat deficiency of the eight extraordinary meridians; second, it can replenish the qi in the lungs. When the lung qi is strong, the qi of the four internal organs is strong, the essence is naturally generated, and the body is naturally strong; third, it can replenish qi and dredge the blood vessels, promote qi circulation to lead blood, replenish qi and blood, and dredge the blood vessels. The extraordinary meridians can be replenished, the qi and blood of the twelve meridians are accumulated, and the skin and muscles are nourished, thereby achieving the function of strengthening the vital energy and curing impotence.

[0023] Cistanche deserticola has a sweet, warm, and rich flavor, and enters the kidney and large intestine meridians. It nourishes kidney essence and nourishes and builds muscle. This herb is warm without being dry, and its sweet and salty properties nourish and replenish, replenishing fire and supporting yang without damaging yin. It is effective for treating the five labors and seven injuries, as well as kidney deficiency and emaciation. It is an excellent remedy for replenishing kidney essence and building muscle. When used in yin-nourishing tonics, Cistanche deserticola can nourish yang and replenish yin, replenishing essence and blood, and promoting muscle growth and warming the waist and knees, thereby improving symptoms of paralysis, such as chills, cold limbs, and decreased muscle strength. Cistanche deserticola nourishes the kidney essence, warms and replenishes essence, nourishes and builds muscle. When used in combination with ginseng, the main herb, it can replenish essence and marrow, tonify qi, nourish blood, and enhance yang.

[0024] In summary, this prescription selects ginseng to support the true essence and nourish the Du channel, adhering to the principle of "those who are deficient in form should be warmed with qi"; it selects Cistanche deserticola to replenish the true essence and nourish the muscles, adhering to the principle of "those who are deficient in essence should be supplemented with flavor", so as to achieve the purpose of supporting the true essence and relieving atrophy, and nourishing the muscles.

[0025] The process of formulating this prescription not only reflects the guidance of traditional Chinese medicine theory on clinical prescription composition, which is based on the deepening of clinical human experience data, but also reflects the organic combination of modern bioinformatics technology and experimental technology.

[0026] The pharmaceutical composition of the present invention is mainly used to treat the Du Yuan deficiency syndrome of amyotrophic lateral sclerosis, which is characterized by decreased muscle strength, muscle atrophy, muscle twitching, muscle stiffness or weakness, unclear articulation, dysphagia, shortness of breath, soreness of the waist and knees, fatigue and laziness, fear of cold and cold limbs, pale tongue, and deep and weak pulse. If the true essence is insufficient, the function of Yang to be active and uplifting will be reduced, and the active parts of the body will be unable to move, manifested as limb weakness, difficulty walking, fatigue and laziness, shortness of breath, etc. If the whole body's primordial Yang and vital energy are insufficient, there is no way to nourish the muscles and skin, resulting in muscle weakness and atrophy. If the Yang Qi is deficient, the tendons and veins will not be able to be warmed and nourished, and symptoms such as increased muscle tone and muscle stiffness can be seen. If the true essence is insufficient, the clear Yang will not rise, the turbid Yin will not descend, the Qi mechanism will be disordered, and dysphagia will occur if the pharyngeal gate is not functioning properly. Malnutrition of the Du channel can lead to neck weakness, even lumbar weakness, and lower back and knee pain. A lack of Du channel replenishment prevents the ascending of clear yang, leading to dysfunction of the upper orifices, which can cause slurred speech, dysphagia, and coughing when drinking water. Based on this, the author innovatively proposed the pathogenesis of ALS as "insufficient true essence and malnutrition of the Du channel," emphasizing that insufficient true essence is the root cause of the disease and malnutrition of the Du channel is the key to the pathogenesis. This established the treatment approach of "strengthening the vital essence to heal atrophy and nourishing the flesh to regenerate muscle," and formulated the formulation of the pharmaceutical composition of this invention.

[0027] The pharmaceutical composition of the present invention is formulated to fully address the pathogenesis of the disease. Ginseng is selected to greatly invigorate vital energy, nourish the Qi and collaterals, and strengthen the vital energy to cure impotence. It is combined with Cistanche deserticola, which nourishes and replenishes the true essence. The composition can not only nourish the kidney and replenish the essence and blood, but also promote the generation of yang and replenish yin in a yin-tonifying agent. The combination of supporting vital energy and invigorating the kidney and replenishing the essence achieves the purpose of strengthening the vital energy to cure impotence, nourishing the vital energy and regenerating the flesh. The prescription is an effective prescription formed under the guidance of traditional Chinese medicine theory on clinical prescription, based on the deepening of clinical human experience data, and the organic combination of modern bioinformatics and experimental technology. The etiology and pathogenesis, treatment principles and methods, prescription and medication, theory, method, prescription, and medication are consistent, with a high degree of fit and a reasonable prescription.

[0028] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0029] The invention relates to a traditional Chinese medicine composition for treating amyotrophic lateral sclerosis. The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 3-9 parts of ginseng and 3-9 parts of cistanche deserticola.

[0030] Furthermore, the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 4-6 parts of ginseng and 4-6 parts of Cistanche deserticola.

[0031] Furthermore, the Chinese medicine composition can also be composed of the following raw materials in parts by weight: 3 parts of ginseng and 9 parts of Cistanche deserticola.

[0032] Furthermore, the Chinese medicine composition can also be composed of the following raw materials in parts by weight: 6 parts of ginseng and 6 parts of Cistanche deserticola.

[0033] The traditional Chinese medicine composition is further composed of the following raw materials in parts by weight: 9 parts of ginseng and 3 parts of Cistanche deserticola.

[0034] The traditional Chinese medicine composition is further composed of the following raw materials in parts by weight: 7 parts of ginseng and 4 parts of Cistanche deserticola.

[0035] The present invention also provides a preparation method of the traditional Chinese medicine composition, wherein the active ingredient is prepared by the following steps: weighing ginseng and cistanche deserticola according to the prescribed amount, decocting with water, filtering, and concentrating the filtrate under reduced pressure to obtain an extract, which is the active ingredient of the traditional Chinese medicine composition.

[0036] The present invention also provides the use of the traditional Chinese medicine composition in preparing a treatment for the symptoms of amyotrophic lateral sclerosis manifested by decreased muscle strength, muscle atrophy, muscle twitching, muscle stiffness or weakness, slurred speech, difficulty swallowing, shortness of breath, soreness of waist and knees, fatigue and laziness, or chills and cold limbs.

[0037] The present invention also provides the Chinese medicine composition, which is used in preparing medicines for delaying the paralysis time of amyotrophic lateral sclerosis, improving damaged nerve function, alleviating sciatica, and increasing the co-localization degree of neuromuscular junctions.

[0038] The present invention also provides the use of the traditional Chinese medicine composition in preparing a medicine for prolonging the hanging time of mice, increasing the peak value of muscle action potential and shortening the selection time course.

[0039] The present invention also provides the use of the traditional Chinese medicine composition in preparing a medicine for improving the symptoms of demyelination in the central nervous system, improving muscle strength and muscle fiber fullness, and increasing the blood supply to muscle tissue.

[0040] The present invention also provides the use of the traditional Chinese medicine composition in preparing a drug for treating sarcopenia.

[0041] The present invention also provides the Chinese medicine composition, which is used in the preparation of a drug for improving neuroelectrophysiology, reducing serum malondialdehyde content, promoting axonal growth and myelin regeneration, and protecting nerve function; increasing the co-localization degree of muscle presynaptic markers and acetylcholine receptor markers, increasing the number of positive gastrocnemius satellite cells and myelin marker (MBP), improving the integrity of the neuromuscular junction structure, increasing the expression of neurite markers (Tuj-1) and synaptic vesicle protein (SV2A), promoting myelin regeneration, improving behavioral changes caused by demyelination, improving signal transmission from motor neurons to muscles, and delaying the progression of amyotrophic lateral sclerosis.

[0042] Furthermore, the present invention provides a medicine comprising the aforementioned traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the aforementioned preparation method.

[0043] The dosage form of the traditional Chinese medicine composition protected by the present invention is capsule, tablet, granule, injection, pill, powder, spray or oral liquid.

[0044] In order to realize the above dosage forms, pharmaceutically acceptable excipients need to be added when preparing these dosage forms, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavorings include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, insect wax, etc.

[0045] Furthermore, the granules of the Chinese medicine composition of the present invention are prepared by the following steps:

[0046] (1) Weigh ginseng and Cistanche deserticola according to the prescribed amount, add water and boil until complete, filter the water extract, and concentrate the filtrate to an extract for later use;

[0047] (2) Take the above extract, spray dry it using a spray dryer, collect the spray powder and set aside;

[0048] (3) Add appropriate amount of auxiliary materials to the spray powder obtained in step (2) and granulate it into granules according to conventional methods.

[0049] Furthermore, the preparation method of the granules of the Chinese medicine composition of the present invention is:

[0050] (1) Weigh ginseng and Cistanche deserticola according to the prescribed amount, add water and boil three times, add 10 times the amount of water for the first time, and add 8 times the amount of water for the second and third times, boil for 1.5 hours each time, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.05-1.10 (60℃) for later use;

[0051] (2) Take the above extract, spray dry it using a spray dryer, collect the spray powder and set aside;

[0052] (3) Add appropriate amount of auxiliary materials to the spray powder obtained in step (2) and granulate it into granules according to conventional methods.

[0053] Furthermore, the present invention also provides a method for treating a disease, which comprises administering the aforementioned traditional Chinese medicine composition, or administering the traditional Chinese medicine composition prepared by the aforementioned preparation method, or administering the aforementioned medicine.

[0054] The treatment includes at least one of the following:

[0055] Delay the paralysis time of amyotrophic lateral sclerosis, improve damaged nerve function, alleviate sciatica, and increase the colocalization degree of neuromuscular junction;

[0056] Prolonged the hanging time of mice, increased the peak value of muscle action potential and shortened the selection time course;

[0057] Improve the symptoms of demyelination in the central nervous system, increase muscle strength and muscle fiber fullness, and increase blood supply to muscle tissue;

[0058] Improve neuroelectrophysiology, reduce serum malondialdehyde content, promote axonal growth and myelin regeneration, protect nerve function, increase the co-localization of muscle presynaptic markers and acetylcholine receptor markers, increase the number of positive gastrocnemius satellite cells and myelin marker (MBP), improve the integrity of neuromuscular junction structure, increase the expression of neurite markers (Tuj-1) and synaptic vesicle protein (SV2A), promote myelin regeneration, improve behavioral changes caused by demyelination or improve signal transmission from motor neurons to muscles, and delay the progression of amyotrophic lateral sclerosis.

[0059] The method for preparing the active ingredients of the traditional Chinese medicine composition provided by the present invention has simple process, convenient operation, safety and environmental protection, and can retain the active ingredients in the traditional Chinese medicine to the greatest extent, thereby ensuring the efficacy of the medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 EC50 of TN-2242 series compounds in glutamate injury model;

[0061] Figure 2 Survival rate of TN-2242 series compounds in glutamate injury model (*P<0.05);

[0062] Figure 3 Survival curves - regardless of sex;

[0063] Fig. 4 Survival curves - female;

[0064] Figure 5. HE staining of soleus muscle of blank control group (female) 20×;

[0065] Figure 6. HE staining of soleus muscle of blank control group (male) 20×;

[0066] Figure 7 HE staining of soleus muscle in the model control group (female) 20×;

[0067] Figure 8 HE staining of soleus muscle in the model control group (male) 20×;

[0068] Figure 9 HE staining of soleus muscle in the positive control group (female) 20×;

[0069] Figure 10 HE staining of soleus muscle in the positive control group (female) 20×;

[0070] Figure 11 HE staining of soleus muscle of the low-dose group (female) of the pharmaceutical composition of the present invention (20×);

[0071] Figure 12 HE staining of soleus muscle (male) in the low-dose group of the pharmaceutical composition of the present invention (20×);

[0072] Figure 13 HE staining of soleus muscle (female) in the dosage group of the pharmaceutical composition of the present invention (20×);

[0073] Figure 14 HE staining of soleus muscle (male) in the dosage group of the pharmaceutical composition of the present invention (20×);

[0074] Figure 15 HE staining of soleus muscle of the high-dose group (female) of the pharmaceutical composition of the present invention (20×);

[0075] Figure 16 HE staining of soleus muscle (male) in the high-dose group of the pharmaceutical composition of the present invention (20×);

[0076] Figure 17 Blank control group (female) gastrocnemius muscle HE staining 10×;

[0077] Figure 18 HE staining of gastrocnemius muscle of blank control group (male) 10×;

[0078] Figure 19 HE staining of gastrocnemius muscle in model control group (female) 10×;

[0079] Figure 20 HE staining of gastrocnemius muscle in model control group (male) 10×;

[0080] Figure 21. Co-staining immunofluorescence of a presynaptic marker (synaptotagmin-2) and an acetylcholine receptor (nAChR) marker (α-BTX) in the gastrocnemius muscle;

[0081] Figure 22 shows the results of co-staining immunofluorescence of gastrocnemius muscle satellite cell marker (Pax7) and laminin 2α;

[0082] Figure 23 Immunofluorescence of myelin basic protein (MBP) in the spinal cord;

[0083] Figure 24 Immunofluorescence of neurite marker (Tuj-1) in the spinal cord. DETAILED DESCRIPTION

[0084] The present invention will be further described in detail below with reference to specific embodiments.

[0085] Example 1

[0086] Prescription: 600g ginseng, 600g Cistanche deserticola

[0087] Preparation method:

[0088] Weigh ginseng and Cistanche deserticola according to the prescribed amount, add water and boil three times, adding 10 times the amount of water for the first time, and 8 times the amount of water for the second and third times. Boil for 1.5 hours each time, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.05-1.10 (60°C), filter, and set aside. Take the above extract and spray dry it in a spray dryer with an air inlet temperature of 175°C ± 5°C and an air outlet temperature of 100°C ± 5°C. Collect the spray powder and set aside. Mix the spray powder and maltodextrin and granulate to obtain granules, set aside. Take the above granules and package them in 5g bags.

[0089] Example 2

[0090] Prescription: 300g ginseng, 900g Cistanche deserticola

[0091] Preparation method:

[0092] Weigh ginseng and Cistanche deserticola according to the prescribed amount and decoct twice with water, adding 10 times the amount of water the first time and 8 times the amount the second time. Decoct for 1.5 hours each time, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.05-1.10 (60°C). Filter and set aside. Spray dry the extract in a spray dryer at an inlet temperature of 175°C ± 5°C and an outlet temperature of 100°C ± 5°C. Collect the spray powder and set aside. Mix the spray powder and maltodextrin, granulate, and encapsulate.

[0093] Example 3

[0094] Prescription: 900g ginseng, 300g Cistanche deserticola

[0095] Preparation method:

[0096] Weigh ginseng and Cistanche deserticola according to the prescribed amount and decoct three times with water, adding 8 times the amount of water for the first decoction and 10 times the amount of water for the second and third decoctions. Decoction for 2 hours each time, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.05-1.10 (60°C). Filter and set aside. Take the extract and spray dry it in a spray dryer at an inlet air temperature of 175°C ± 5°C and an outlet air temperature of 100°C ± 5°C. Collect the spray powder and set aside. Mix the spray powder and maltodextrin, granulate, and prepare tablets according to conventional methods.

[0097] Example 4

[0098] Prescription: 700g ginseng, 400g Cistanche deserticola

[0099] Preparation method:

[0100] Weigh ginseng and Cistanche deserticola according to the prescribed amount, add water and boil twice, adding 10 times the amount of water each time, boiling for 2 hours each time, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.05-1.10 (60°C), filter, and prepare an oral solution according to conventional methods.

[0101] Example 5

[0102] 600g ginseng, 600g cistanche deserticola

[0103] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0104] Comparative Example 1

[0105] 1200g ginseng

[0106] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0107] Comparative Example 2

[0108] Cistanche deserticola 1200g

[0109] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0110] Comparative Example 3

[0111] 860g ginseng, 340g epimedium

[0112] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0113] Comparative Example 4

[0114] Ginseng 600g Epimedium 600g

[0115] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0116] Comparative Example 5

[0117] 1000g ginseng, 200g cistanche deserticola

[0118] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0119] Comparative Example 6

[0120] 200g ginseng, 1000g cistanche

[0121] Add 10 times the amount of water and boil twice, filter, concentrate under reduced pressure to become extract, and dry spray powder.

[0122] Experimental Example 1: Study on the intervention effects of multiple extracts and their different combinations on ALS-related cell models

[0123] 1. Purpose of the experiment

[0124] In this study, glutamate-damaged rat pheochromoma cells PC-12 were used as carriers to determine the intervention effects of various extracts and different compatibility groups on the survival activity of the glutamate-damaged PC-12 cell model, and to determine the extracts or compatibility groups for subsequent studies.

[0125] 2 Experimental Materials

[0126] 2.1 Samples to be screened

[0127] 2.2 Reagents and tools

[0128] 2.2.1 Modeling drug: L-glutamic acid

[0129] 2.2.2 Reagents and kits: DMEM culture medium, DMSO, trypsin, double antibody, fetal bovine serum, PBS, MTS kit

[0130] 2.3 Experimental system

[0131] 2.3.1 Cell line name and source

[0132] Rat pheochromoma cells PC-12: cryopreserved in our cell laboratory.

[0133] 2.3.2 Instrument system

[0134] SW-CJ-2FD clean bench, Suzhou Antai Air Technology Co., Ltd.

[0135] Protect-2FD-S clean bench, Thermo Fisher Scientific (China) Co., Ltd.

[0136] Olympus CKX53 microscope, Olympus Corporation.

[0137] Thermo3111 CO2 incubator, Thermo Fisher Scientific Inc.

[0138] Infinite M200PRO microplate reader, TECAN, Switzerland.

[0139] AMQAX1000 cell counter, Thermo Fisher Scientific Inc.

[0140] DK-98-Ⅱ Electric Constant Temperature Water Bath, Tianjin Test Instrument Co., Ltd.

[0141] Sorvall ST 16 / 16R high-speed refrigerated centrifuge, Thermo Fisher Scientific Inc.

[0142] BPN-CH CO2 incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.

[0143] 3. Test methods

[0144] 3.1 Preparation of main reagents:

[0145] Each extract and its compatibility components were dissolved in DMSO to prepare a 500 mg / ml stock solution and stored at -20°C for later use.

[0146] 3.2 Experimental groups and intervention methods:

[0147] Cells in logarithmic growth phase were taken and inoculated into cell culture plates (cell density 1×10 5 When the cells reached 80% fusion, the old culture medium was discarded and the cells were divided into the following groups (6 replicate wells were set up in parallel for each group):

[0148] 3.2.1 Experimental grouping of samples to be screened

[0149] (1) Blank control group (Control): Continue culturing with serum-free culture medium.

[0150] (2) Model group: cells were cultured in serum-free medium containing 20 mM glutamate.

[0151] (3) Solvent group (DMSO): Serum-free culture medium containing 0.1% DMSO and 20 mM glutamate was added and culture was continued.

[0152] (4) Drug group: Add 500 μg / ml, 50 μg / ml, 5 μg / ml and other concentrations of each drug and 20 mM glutamate in serum-free medium for culture.

[0153] 4.Indicator detection

[0154] Survival activity

[0155] The viability assay was performed in a 96-well plate using MTS detection reagents, and the operation steps were strictly in accordance with the kit instructions.

[0156] 5 Statistical methods

[0157] The experimental data are expressed as mean ± standard deviation said that EC 50 The data were calculated using GraphPad Prism 8 software and analyzed using T-TEST.

[0158] 6. Results

[0159] TN-2242 series compounds were added to glutamate-damaged PC12 cells in the order of 500 μg / ml, 50 μg / ml, 5 μg / ml, 500 ng / ml, 50 ng / ml, and 5 ng / ml. After 24 h, the cell survival rate was detected by MTS and the EC value of each compound for inhibiting glutamate-induced neuronal damage was calculated.50 The results are shown in Figure 1. 50 The lowest value was 1.225μg / ml. The other extracts and the combination group EC 50 See Table 1 for values.

[0160] Table 1 EC values ​​of TN-2242 series compounds in glutamate injury model 50

[0161] Then, the pharmaceutical composition EC of the present invention is used 50 The protective effects of the extracts and the combination groups on PC12 cells damaged by glutamate were tested at a concentration of 1 μg / ml. The results showed ( FIG. 2 , Table 2 ) that the pharmaceutical composition of the present invention was superior to comparative examples 1-6.

[0162] Table 2 Survival rate of TN-2242 series compounds in glutamate injury model

[0163] The above test results show that the protective effect of the pharmaceutical composition of the present invention in the glutamate injury model is better than that of other extracts and compatibility groups.

[0164] 7. Recordkeeping

[0165] Except for the data directly collected by computers or automated instruments, all other data generated in the actual research are recorded on forms or recording papers and organized and bound at any time.

[0166] 8. Data Archiving

[0167] 8.1 Depository: New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd.

[0168] 8.2 Address: No. 238, Tianshan Street, Shijiazhuang High-tech Development Zone

[0169] Experimental Example 2: Effect of the pharmaceutical composition of the present invention on the survival time of amyotrophic lateral sclerosis SOD1-G93A transgenic mice

[0170] Purpose of the trial

[0171] The therapeutic effect of the pharmaceutical composition of the present invention on amyotrophic lateral sclerosis was observed using SOD1-G93A transgenic mice.

[0172] 1 Experimental Materials

[0173] 1.1 Test sample

[0174] 1.1.1 Name: Pharmaceutical composition of the present invention, prepared according to Example 1, abbreviation: TMC-073. Sample number: GLPTN-2106.

[0175] 1.1.2 Properties: This product is a yellow-brown powder with a slight fragrance and a slightly bitter taste.

[0176] 1.1.3 Function and indications: Amyotrophic lateral sclerosis

[0177] 1.1.4 Clinical dosage and route of administration: 18 g crude drug / day, orally.

[0178] 1.1.5 Specifications: 3.1g crude drug / g powder.

[0179] 1.1.6 Source and batch number: Shijiazhuang Yiling Pharmaceutical Co., Ltd., 210701.

[0180] 1.1.7 Storage conditions: Keep in a dry place at room temperature.

[0181] 1.1.8 Validity period: tentatively set to July 27, 2022.

[0182] 1.2 Positive drugs and main reagents

[0183] 1.2.1 Positive Drug: Riluzole (trade name: Riluzole), 50 mg / tablet, is used to treat amyotrophic lateral sclerosis (ALS), and can prolong patient survival and / or delay tracheotomy. Adults are prescribed 50 mg (1 tablet) orally twice daily. Produced by Sanofi Winthrop Industrie and packaged by Sanofi (Beijing) Pharmaceutical Co., Ltd., batch number: 0KP4C, expiration date: May 2023.

[0184] 1.2.2 Main Reagents: Sterile Water for Injection, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number 1911143205, valid until November 13, 2021. Batch number 2007023202, valid until July 1, 2022.

[0185] 1.3 Experimental System

[0186] 1.3.1 Animal strain: B6SJL-Tg(SOD1-G93A)1Gur / J (Stock Number: 002726) was provided by The Jackson Laboratory, USA. Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. maintained and bred the strain, and identified the offspring of SOD1-G93A transgenic mice. Mice from the same littermates that did not carry the human mutant SOD1 gene served as negative controls.

[0187] 1.3.2 Animal grade: SPF grade.

[0188] 1.3.3 Animal sex and quantity: 150 B6SJL-Tg(SOD1-G93A)1Gur / J mice; 20 mice of the same litter without the human mutant SOD1 gene, both male and female were used.

[0189] 1.3.4 Animal age: 53 days old.

[0190] 1.3.5 Animal weight: Male mice: 22.8 g - 34.4 g, female mice: 16.9 g - 25.3 g.

[0191] 1.3.6 Animal source: Purchased from The Jackson Laboratory in the United States and reproduced by Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0192] 1.3.7 Animal certificate number, issuing unit and receiving date: Certificate number: 320727210100625138, License number: SCXK(Su)201-0008, Issuing unit: Jiangsu Jicui Yakang Biotechnology Co., Ltd., Receiving date: August 11, 2021.

[0193] 1.3.8 Feeding conditions: Mice were caged and raised in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, License number: SYXK(Ji)2020-006. Lighting: 12 hours per day, Temperature: 20 - 26 °C, Relative humidity: 40 - 70%.

[0194] 1.3.9 Quarantine process: New animals were quarantined for 5 days. During this period, it was observed whether the animals drank water and ate normally, and whether their health conditions were good, without signs of disease and death.

[0195] 1.3.10 Feed: SPF mouse and rat maintenance feed, which is intended to be provided by Beijing Keao Xieli Feed Co., Ltd. Feed production license number: Jing Si Zheng(2018)06073, Experimental animal production license number: SCXK(Jing)2019-0003. Product batch number: 21033213, Expiry date: August 31, 2021; Product batch number: 21073213, Expiry date: January 3, 2022; Product batch number: 21093213, Expiry date: March 3, 2022

[0196] 1.3.11 Drinking water: Drink clean water prepared by the ROB-B experimental animal drinking water system. The drinking water bottles were filled for free drinking. The drinking water was tested for all items once a year, mainly including microbial indicators, toxicological indicators, sensory properties and general chemical indicators, and entrusted to a qualified institution for testing; Microbiological limit tests were carried out once every 2 months, entrusted to the Quality Inspection Section of Shijiazhuang Yiling Pharmaceutical Co., Ltd., and the test reports were kept by the animal room and archived annually.

[0197] 1.3.12 Bedding: Ordinary grade corn cob bedding, which shall be sterilized at high temperature and high pressure before use. It is to be provided by Beijing Keao Xieli Feed Co., Ltd., batch number: 21099811.

[0198] 1.3.13 Identification: Mouse ear tags are used for labeling. Ear tag manufacturer: Chuangbo Global (Beijing) Biotechnology Co., Ltd.

[0199] 2 Test methods

[0200] 2.1 Basis of experimental design

[0201] 2.1.1 Adopted standards: Determined by the "Registration Classification and Application Document Requirements for Traditional Chinese Medicine and Natural Medicines" and "Good Manufacturing Practice for Non-clinical Drug Research" issued by the State Food and Drug Administration and related literature.

[0202] 2.1.2 Experimental System Selection: Transgenic mice carrying a human superoxide dismutase (SOD) gene mutation (B6SJL-Tg(SOD1-G93A)1Gur / J) are currently considered the most commonly used and best-performing animal model for amyotrophic lateral sclerosis (ALS). While the pathogenesis of ALS remains unclear, with 90% of cases occurring sporadically and 10% occurring in familial forms (FALS), the discovery of a missense mutation in copper-zinc superoxide dismutase (SOD1) by Rosen in 1993 marked a major milestone in ALS research. The discovery of SOD1 mutations in FALS led to the subsequent development of animal models for ALS. Currently, the SOD1-G93A transgenic mouse for ALS is widely used in both clinical and basic research. This transgenic mouse carries a high copy number of the human SOD1 mutation. Homozygous mice exhibit a reduction in spinal motor neurons and gradually develop neuromuscular dysfunction, including tail-hanging limb tremors and hemiplegia of one or more limbs, around three months after birth. With a lifespan of approximately four to five months, it mimics the chronic, progressive course of ALS and some of its pathological and biochemical manifestations.

[0203] 2.2 Dosage and Grouping

[0204] Based on literature and previous experience, combined with behavioral testing results, ALS mice were randomly divided by body weight and administered for observation after significant changes compared to negative control mice. Specifically, they were divided into: a model control group (M), a positive drug control group (riluzole 0.02g / kg, R), and low-dose (073-L), medium-dose (073-M), and high-dose (073-H) groups of the present invention's pharmaceutical composition, with 30 animals in each group. A negative control group (C, 20 animals total) consisting of wild-type littermate mice that do not carry the human mutant SOD1 gene was also included. Specific experimental groupings and dosages are shown in Appendix 3.

[0205] 2.3 Administration

[0206] The drug is administered by oral gavage, which is consistent with the clinically recommended oral route.

[0207] 2.4 Preparation and storage of test samples

[0208] The clinical kilogram dose of riluzole, calculated based on a body weight of 60 kg, is approximately 0.002 g / kg. The dose administered to mice is equivalent to 10 times the clinical dose, approximately 0.02 g / kg, with a dosing volume of 10 ml / kg and a dosing concentration of 0.02 g / 10 ml. The drug is prepared by grinding the powder from the tablets. Each tablet contains 50 mg of riluzole. For preparation, one tablet is taken and ground to a volume of 25 ml with sterile water for injection.

[0209] The proposed clinical dosage of the pharmaceutical composition of this invention is 18 g of crude drug per day. This trial employed three doses: low, medium, and high: 1.5, 3.0, and 6.0 g of crude drug per kg (equivalent to 5, 10, and 20 times the clinical dosage, respectively). See Table 3 for details. Using sterile water for injection as the solvent, the high, medium, and low doses were prepared, and appropriate amounts of the drug were weighed and ground.

[0210] Appendix 3: Trial Grouping and Dosage Setting

[0211] 2.5 Administration of test products

[0212] The corresponding drug solution was drawn up with a syringe for oral administration, with a dosage of 10 ml / kg. The model control group and the negative control group were given the corresponding volume of sterile water for injection; the dosage of each animal was adjusted weekly according to the most recent body weight.

[0213] 2.6 Determination of model mice

[0214] 2.6.1 Tail suspension test: Observe the limb extension reflex. When the tail of a normal mouse is lifted into the air, the hind limbs of both sides can be naturally extended. When the tail of a mouse is affected by motor neuron disease, the hind limbs can be flexed after the tail is lifted. The scoring criteria are as follows:

[0215] 0 points: No flexion of the hind limbs.

[0216] 1 point: One hind limb is flexed, but the other limb is not flexed.

[0217] Score 2: Bilateral hindlimb flexion is visible. Mice with a score of 2 are considered to be in the symptomatic stage. The tail suspension test is stopped when all ALS mice have a score of 2.

[0218] 2.6.2 Hanging test: Place the mouse on a 3-mesh sieve and quickly turn it over. The time is counted after the mouse is hung upside down on the sieve, with a limit of 90 seconds. Mice that are less than 90 seconds and the actual time are recorded. When the ALS mouse dies, the hanging test is stopped. ALS mice show progressive loss of motor function, manifested by hind limb flexion when the tail is lifted. The degree of abnormality is consistent with the degree of muscle atrophy and loss of muscle strength. This experiment determines whether the transgenic mice have signs of disease based on the results of the tail suspension test. At the same time, combined with literature research and previous experimental experience, the tail suspension and hanging test observations will continue to be performed on each mouse.

[0219] 2.7 Observation indicators, time and content

[0220] 2.7.1 General observation and evaluation methods

[0221] During the experiment, the animals' mental state, disease progression, and survival were observed. The birth date, dosing start time, weight, and death time of each animal were recorded, and the average survival time and cumulative mortality of animals at different times after dosing were calculated.

[0222] Method for determining the death endpoint: From an ethical perspective, according to the literature [4] Mice were considered dead if they could not turn over to a normal position on their side after lying on a flat surface for 25 seconds.

[0223] 2.7.2 Behavioral indicators

[0224] During the trial administration phase, neurological function evaluation (Snow scoring method, twice a week) was performed. The specific criteria for neurological scoring were as follows:

[0225] 1.0 points: abnormal hind limb extension or tremor when the mouse is suspended;

[0226] 1.5 points - weakness of one hind limb;

[0227] 2.0 points - bilateral hind limb weakness and abnormal gait;

[0228] 2.5 points - obvious weakness on one side of the limb;

[0229] 3.0 points - obvious weakness of bilateral limbs;

[0230] 3.5 points--paralysis of one hind limb;

[0231] 4.0 points - bilateral hind limb paralysis;

[0232] 5.0 points: The mouse cannot turn over within 20 seconds after being placed in supine position.

[0233] The time from drug administration to the first appearance of a neurological function score ≥ 3.5 was used as the onset time of paralysis. The average onset time of paralysis symptoms in animals, the percentage of animals with paralysis at different times after drug administration, and the paralysis delay rate of each drug group were calculated. The delay rate calculation formula is as follows:

[0234] 2.7.3 Criteria for mouse culling

[0235] During the experiment, the general health status and motor function changes of the mice were observed. If death occurred outside the regular onset period or death did not meet the pattern of progressive motor function decline of ALS disease, it was considered as non-ALS death; no onset changes were observed according to the ALS model judgment criteria; the measurement data of such mice were eliminated and did not participate in the final data statistical analysis.

[0236] 2.8 Notification to relevant staff

[0237] Notify the animal room when purchasing animals, and notify the pathology room for treatment when animals show abnormal conditions.

[0238] 2.9 Statistical Methods

[0239] The experimental data are expressed as mean ± standard deviation Statistical analysis was performed using SPSS 25.0 software. Survival analysis was performed using the Log Rank and Tarone-Ware tests, and survival curves were plotted using Graphpad Prism 8.0. Paralysis duration and behavioral scores were tested for normality. For data that conformed to a normal distribution, means were compared using one-way analysis of variance. If the variances were equal, pairwise comparisons were performed using the least significant difference method. If the variances were unequal, Dunnett's T3 test was used. If the data did not conform to a normal distribution, nonparametric tests were used for statistical analysis.

[0240] 2.10 Instrument System

[0241] JJ2000 electronic balance, Changshu Shuangjie Testing Instrument Factory.

[0242] Sartorius BSA2201 electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0243] BT224S Sartorius precision analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0244] 3 Results

[0245] 3.1 Impact on survival time

[0246] As shown in Appendix 4 and Figure 3, there were no significant differences in survival time between the treatment groups and the model group (P>0.05). The mean survival days were slightly prolonged in all treatment groups. Survival time analysis of the animals by sex is detailed in Figure 4: The mean survival days of female mice at both the low and high doses were significantly prolonged compared to the model group; however, there was no difference in the mean survival days of male mice compared to the model group.

[0247] Schedule 4 Survival Time Note: Compared with the model group: *P<0.05.

[0248] 3.2 Impact on paralysis duration

[0249] The time from dosing to the first neurological function score of 3.5 or higher was used as the time of paralysis onset, and the average time of paralysis onset was calculated. If an animal died without developing a paralysis score, the time of death was used as the time of paralysis onset to ensure the number of animals analyzed. The results are shown in Appendix 5. The positive control group and all dosing groups significantly delayed the time of paralysis onset (P < 0.05). Analysis of the time of paralysis onset by sex showed that both the medium- and high-dose groups significantly delayed the onset of paralysis in both male and female mice.

[0250] Schedule 5 Paralysis Time Note: Compared with the model group: *P<0.05, **P<0.01.

[0251] 3.3 Effects on hanging time and neurological function scores

[0252] As shown in Appendix 6 and Appendix 7, the neurological impairment in the medium-dose group at D39 and D46, and in the high-dose group at D31 and D34, was significantly reduced compared with the model group (P < 0.05). The suspension time in the positive control group and the high-dose group at D46 was significantly prolonged compared with the model group (P < 0.05).

[0253] Appendix 6 Neurological function score Note: Compared with the model group: *P<0.05.

[0254] Schedule 7 Hanging Time Note: Compared with the model group: *P<0.05.

[0255] 4 Conclusion

[0256] The study used SOD1-G93A transgenic mice, a classic model of ALS, and began administering the drug at 70 days of age to evaluate the therapeutic effects of different doses of the pharmaceutical composition of the present invention. The results showed that all dose groups of the test drug delayed the onset of paralysis. The medium and high dose groups (3.0 g and 6.0 g of the crude drug / kg) improved the impaired neurological function of SOD1-G93A transgenic mice to varying degrees, while the high dose group (6.0 g of the crude drug / kg) prolonged the mice's hanging time. No significant therapeutic effect was observed in any of the drug groups on prolonging survival.

[0257] 5. Recordkeeping

[0258] In addition to data collected directly by computers or automated instruments, all other data generated during the actual research process must be recorded on spreadsheets or charts and organized and bound at all times. All data records must be signed by the operator and verifier, and the date of recording must be noted. When the final report is submitted, all original data, documents, and other relevant materials must be handed over to the data archive for safekeeping.

[0259] Test Example 3: Effect of the pharmaceutical composition of the present invention on skeletal muscle disuse atrophy in hindlimb-suspension mice

[0260] Purpose of the trial

[0261] A muscle atrophy model was established by suspending the hind limbs of mice to simulate clinical sarcopenia, and whether the pharmaceutical composition of the present invention has protective and therapeutic effects on skeletal muscle atrophy was observed.

[0262] 1 Experimental Materials

[0263] 1.1 Test sample: The pharmaceutical composition of the present invention, prepared as in Test Example 2, abbreviated as TMC-073.

[0264] 1.2 Positive control and main reagents

[0265] 1.2.1 Positive Control: Whey protein powder, manufactured by Field Pharmaceuticals, Inc., USA. Store in a cool, dry place away from direct sunlight. Sample number: GLPTN-2106-D2, batch number: 03172023B, valid until March 17, 2023.

[0266] 1.2.2 Main reagents:

[0267] Sterile water for injection, Shijiazhuang Siyao Co., Ltd., batch number 2009053201, valid until September 4, 2022.

[0268] 10% neutral buffered formalin fixative, Beijing Yili Fine Chemicals Co., Ltd., batch number 20220406, valid until 2023.04.05.

[0269] Isoflurane: Beijing Youcheng Jiaye Biotechnology Co., Ltd., batch number G45780, expiration date December 30, 2024.

[0270] 1.3 Experimental system

[0271] 1.3.1 Animal strain: C57BL / 6J.

[0272] 1.3.2 Animal level: SPF level.

[0273] 1.3.3 Animal gender and quantity: 100 animals were purchased (50 males and 50 females), and 96 animals were actually used in the experiment.

[0274] 1.3.4 Animal age at the start of dosing: The protocol requires 5 - 7 weeks old, and the actual age is 5 - 7 weeks old.

[0275] 1.3.5 Animal weight at the start of dosing: The protocol requires male mice to be 22 - 24 g and female mice to be 21 - 23 g. The actual weights are: male mice 20.7 - 24.6 g, female mice 16.6 - 19.5 g.

[0276] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0277] 1.3.7 Animal certificate number, issuing unit, and receiving date: Certificate numbers 110011220106187852, 110011220106187965, license number SCXK(Beijing)2021 - 0006, the issuing unit is Beijing Vital River Laboratory Animal Technology Co., Ltd., and the receiving date is June 22, 2022.

[0278] ))7 Animal housing conditions: Mice are caged and housed in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine, license number: SYXK(Hebei)2020 - 006. Lighting is 12 hours per day, temperature is 20 - 26 °C, and relative humidity is 40 - 70%.

[0279] 1.3.9 Quarantine process: The quarantine period for newly arrived animals is 7 days, and no abnormal conditions in animal drinking water, feeding, and health were found during the quarantine period.

[0280] 1.3.10 Feed: SPF mouse and rat maintenance feed, Beijing Keao Xieli Feed Co., Ltd., feed production license number: Beijing Feed License (2018)06073, certificate number: 1112622200083240, batch number: 22043213.

[0281] 1.3.11 Litter: Ordinary - grade corn cob litter, provided by Beijing Keao Xieli Feed Co., Ltd., used after being sterilized by high - temperature and high - pressure, batch number: 22049811.

[0282] 1.3.12 Drinking Water: Clean water is prepared by the ROB-B experimental animal drinking water system and bottled for free drinking.

[0283] 1.3.13 Identification: Use ear tags. Ear tag manufacturer: Chuangbo Global (Beijing) Biotechnology Co., Ltd.

[0284] 2 Test methods

[0285] 2.1 Basis of experimental design

[0286] 2.1.1 Adoption of standards: Determined by the "Classification and Application Material Requirements for Registration of Traditional Chinese Medicines" and "Good Manufacturing Practice for Non-clinical Drug Research" issued by the State Food and Drug Administration and relevant literature.

[0287] 2.1.2 Experimental System Selection: Skeletal muscle is characterized by rapid adaptation to changes in the external environment. Under unloaded conditions such as hypokinesia, immobilization, prolonged bed rest, and weightlessness, it exhibits a decrease in muscle mass and / or strength, or a decrease in muscle physiological function. Disuse-induced muscle atrophy in mice can be demonstrated by hind limb suspension, demonstrating distinct characteristics of sarcopenia. This approach is a useful alternative for simulating human sarcopenia and studying its prevention and treatment.

[0288] 2.1.3 Instructions for the selection of positive control substances: Currently, the main treatments for sarcopenia include drug regimens (mainly testosterone and growth hormone), protein supplementation, resistance training, etc. Drug regimens such as testosterone supplementation and growth hormone supplementation may cause side effects such as fluid retention, prostate cancer, and polycythemia, and have not been widely used in clinical practice. Providing nutritional protein supplements and resistance training are currently commonly used symptomatic treatments in clinical practice. Therefore, whey protein powder was selected as the positive control substance in this experiment. Studies have shown that the adult dosage of whey protein is 0.5-1g / kg.day, which can improve the muscle strength and function of patients with sarcopenia. Therefore, this experiment set the mouse dosage at 10g / kg.day (equivalent to 10 times the clinical dosage).

[0289] 2.2 Dosage and Grouping

[0290] Of 100 mice, 96 were randomly divided into the following groups based on body weight: a blank control group, a model control group, a positive control group (whey protein powder), and a low-dose TCM-073 group (1.50 g crude drug / kg), a medium-dose TCM-073 group (3.00 g crude drug / kg), and a high-dose TCM-073 group (6.00 g crude drug / kg). Each group consisted of 16 animals, half male and half female. Specific experimental groups and doses are shown in Appendix 8.

[0291] Table 8 Trial grouping and dose setting

[0292] 2.3 Model establishment

[0293] Except for the animals in the blank control group, the tails of the mice in the other groups were fixed to the top of the cage using a fixing device (the angle between the mouse trunk and the ground was <30°), allowing the mice to rotate and move 360°. The hind limbs were lifted off the ground to remove the weight to create a hind limb skeletal muscle disuse atrophy model to simulate clinical sarcopenia.

[0294] During the hind limb suspension period, one mouse was placed in each cage for a total of 14 days. Afterwards, the suspension was released, the hind limbs were lowered, and spontaneous recovery was allowed for 9 days.

[0295] 2.4 Administration

[0296] The drug is administered by oral gavage, which is consistent with the clinically recommended oral route.

[0297] 2.5 Preparation and storage of test samples

[0298] The proposed clinical dose of TCM-073 is 18g of crude drug per day. This trial employed three doses: low, medium, and high: 1.50, 3.00, and 6.00g of crude drug per kg (equivalent to 5, 10, and 20 times the clinical dose, respectively). See Table 1 for details. Using sterile water for injection as the solvent, prepare the high, medium, and low doses. Weigh an appropriate amount of drug, grind, and mix thoroughly, then dilute to the desired volume. Prepare immediately before use.

[0299] 2.6 Administration of Test Articles

[0300] The corresponding drug solution was drawn up with a syringe and gavage was performed at a volume of 10 ml / kg. The model control group and the blank control group were given the corresponding volume of sterile water for injection. Drug administration began on the day of hind limb suspension and continued for 23 consecutive days.

[0301] 2.7 Observation indicators, time and content

[0302] 2.7.1 General Observations

[0303] During hindlimb suspension, ensure that the animal can eat and drink only by forelimb movements during the suspension period and whether the animal is in any distress.

[0304] The body weight of the animals was recorded on the 1st and 14th days of suspension, the 7th day of spontaneous recovery and the day of autopsy.

[0305] 2.7.2 Behavioral indicator testing

[0306] The grip strength test was performed on the 13th day after the animals were suspended in the hind limbs and on the 6th day after spontaneous recovery.

[0307] The grip strength tester was placed horizontally so that the grip plate showed horizontal movement. The grip strength of the mouse's hind limbs was measured and recorded. Each mouse was measured three times and the average of the three times was taken to evaluate the effect of the drug on the animal's limb strength.

[0308] 2.7.3 Muscle-related index detection

[0309] 2.7.3.1 Laser Doppler Imaging of Hindlimb Muscle Blood Flow

[0310] On the day after the last administration, mice were anesthetized and placed in a prone position. The skin of the left hind limb was cut open to expose a prominent blood vessel (small saphenous vein) on the surface of the gastrocnemius muscle. The blood flow value of this vessel was recorded for 2 minutes.

[0311] 2.7.3.2 Muscle wet weight

[0312] After the hind limb blood flow test was completed, the mice were killed by cervical dislocation. The gastrocnemius and soleus muscles of the left leg were removed, dried with filter paper, and weighed. The muscle index was calculated according to the following formula:

[0313] Muscle index = muscle weight (g) / body weight (g) × 1000

[0314] 2.7.3.3 Histopathological examination

[0315] The gastrocnemius and soleus muscles of the right leg of mice were dissected and preserved in 10% neutral buffered formalin fixative. HE staining was performed to observe the changes in muscle tissue morphology (gastrocnemius and soleus).

[0316] 2.8 Notification to relevant staff

[0317] Notify the animal room when purchasing animals, and notify the pathology room for treatment when animals show abnormal conditions.

[0318] 2.9 Instrument System

[0319] Sartorius BSA2201 electronic balance, Sartorius (Shanghai) Trading Co., Ltd.

[0320] BT224S Sartorius precision analytical balance, Sartorius (Shanghai) Trading Co., Ltd.

[0321] NVT2201ZH electronic balance, Ohaus Instruments (Shanghai) Co., Ltd.

[0322] BlueSpin standard magnetic stirrer, Dalong Xingchuang Laboratory Instrument (Beijing) Co., Ltd.

[0323] DEF2-002 grip force tester, Chatillon Force Measurement.

[0324] MoorLDI2-HIR Laser Doppler Imaging System, Moor Instruments Ltd.

[0325] R500IE small animal anesthesia machine, Shenzhen Ruiwode Life Science Technology Co., Ltd.

[0326] 2.10 Statistical Methods

[0327] The experimental data are expressed as mean ± standard deviation SPSS 26.0 statistical software was used for statistical analysis. A normality test was first performed. For data that met the normal distribution, one-way analysis of variance was used for mean comparison. If the variances were equal, the least significant difference method was used for pairwise comparisons. If the variances were unequal, Dunnett's T3 test was used. If the data did not meet the normal distribution, non-parametric tests were used for statistical analysis.

[0328] 3 Results

[0329] 3.1 Effect on body weight

[0330] As shown in Appendix 9, during the experiment, there was no significant difference in the body weight of mice in the model control group compared with that in the blank control group, and there was no significant difference in the body weight of mice in the positive control group and each dose group of TCM-073 compared with that in the model control group.

[0331] Table 9 Effect of TCM-073 on body weight of mice with hindlimb suspension-induced muscular atrophy (g)

[0332] 3.2 Effects on hindlimb grip strength

[0333] As shown in Appendix 10, on the 13th day of hindlimb suspension (dose D13), the grip strength test of mice in each group showed that compared with the blank control group, the hindlimb grip strength of mice in the model control group was significantly reduced (p<0.01); the hindlimb grip strength of mice in the other drug-treated groups increased to varying degrees compared with the model control group, with the positive control group and the high-dose TCM-073 group showing significant differences (p<0.05, p<0.01). On the 6th day of spontaneous recovery (dose D20), the grip strength test results showed that the hindlimb grip strength of mice in the model control group was still significantly lower than that of the blank control group (p<0.05). Compared with the model control group, the grip strength of mice in the high-dose TCM-073 group was significantly improved (p<0.05). The grip strength of mice in the other drug-treated groups also increased to varying degrees, but there were no significant differences.

[0334] Table 10 Effects of TCM-073 on hindlimb grip strength in mice with disuse muscular atrophy induced by hindlimb suspension (N) Note: Compared with the blank control group: △ p<0.05, △△ p<0.01; compared with the model control group: *p<0.05, **p<0.01.

[0335] 3.3 Effects on hindlimb muscle blood flow

[0336] As shown in Appendix 11, compared with the blank control group, the hind limb venous blood flow of the mice in the model control group was significantly decreased (p<0.05). Compared with the model control group, the hind limb blood flow of the mice in the positive control group was significantly increased (p<0.05). The hind limb blood flow of the mice in each TCM-073 dose group also increased to varying degrees, among which the increase in the high-dose TCM-073 group was the most significant (p<0.01).

[0337] Table 11 Effects of TCM-073 on blood flow in the left hindlimb of mice with disuse muscular atrophy induced by hindlimb suspension Note: Compared with the blank control group: △ p<0.05; compared with the model control group: *p<0.05, **p<0.01.

[0338] 3.4 Effects on hindlimb muscle weight

[0339] The wet weights of the gastrocnemius and soleus muscles of mice were corrected for body weight and converted into corresponding muscle indices. The results can be seen in Appendix 12. The gastrocnemius index of the mice in the model control group was significantly lower than that in the blank control group (p<0.05). Although the soleus index was lower than that in the blank control group, there was no significant difference. Compared with the model control group, the gastrocnemius index and soleus index of the mice in the positive control group and each dose group of TCM-073 increased to varying degrees, but no significant difference was shown.

[0340] Table 12 Effects of TCM-073 on hindlimb muscle weight index in mice with disuse muscular atrophy induced by hindlimb suspension (mg / g) Note: Compared with the blank control group: △ p<0.05.

[0341] 3.5 Effects on hindlimb muscle histomorphology

[0342] 3.5.1 The pathological examination results of this trial showed that compared with the blank control group, the soleus muscle of the model control group showed slight muscle atrophy (microscopically manifested as reduced muscle fiber volume and visible angular muscle fibers). Compared with the model control group, the soleus muscle lesions of the various drug-treated groups did not show significant improvement; no obvious lesions were found in the gastrocnemius muscles of the model control group and the various drug-treated groups (see Figures 5-20).

[0343] 3.5.2 Effect on Muscle Fiber Cross-Sectional Area

[0344] Muscle histomorphological examination revealed that the soleus muscle of hindlimb-suspended mice exhibited a greater atrophy than the gastrocnemius muscle. The cross-sectional area of ​​the soleus muscle fibers in each group of mice was measured using the digital pathology slide viewing software NDP.view2 (U12388-01). As shown in Appendix 13, compared with the blank control group, the cross-sectional area of ​​the soleus muscle fibers in the model control group was significantly reduced (p < 0.01). Compared with the model control group, the cross-sectional area of ​​the soleus muscle fibers in the positive control group and the high-dose TCM-073 group was significantly increased (p < 0.01). The cross-sectional area of ​​the soleus muscle fibers in the low- and medium-dose TCM-073 groups also increased somewhat, but the differences were not significant.

[0345] Table 13 Effects of TCM-073 on the cross-sectional area of ​​soleus muscle fibers in mice with hindlimb suspension-induced muscular atrophy (μm 2 ) Note: Compared with the blank control group: △△ p<0.01; compared with the model control group: **p<0.01.

[0346] 4 Conclusion

[0347] A sarcopenia model was successfully established in mice by suspending their hind limbs for 14 days. Under these experimental conditions, the pharmaceutical composition of the present invention significantly improved disuse-induced muscle atrophy in mice by increasing muscle strength and muscle fiber fullness, and boosting blood supply to muscle tissue.

[0348] 5 Discussions

[0349] Sarcopenia refers to a syndrome characterized by decreased muscle function and quality due to muscle fiber atrophy and loss. Myofibrosis is a nutritional disorder of skeletal muscle, resulting in a reduction in muscle size due to thinning or even disappearance of muscle fibers. It is often caused by disorders of the muscles themselves or dysfunction of the nervous system, with the main causes including neurogenic, myogenic, disuse, and other causes. Skeletal muscle is constantly degraded and synthesized, and muscle mass is maintained through a balance between the two. When the mechanical load on skeletal muscle continues to decrease, skeletal muscle mass and function decline, leading to disuse muscle atrophy. The most direct change in muscle morphology and structure under disuse is myofibrosis, manifested primarily by decreased muscle mass, decreased volume, reduced cross-sectional area, and changes in muscle fiber type. Changes in muscle function are primarily manifested by decreased muscle strength, altered muscle nerve function, and fatigue resistance.

[0350] This study, by suspending the hind limbs of mice and removing the weight from their hind limbs, altered muscle function and significantly reduced hind limb grip strength. Muscle morphology and structure also underwent significant changes, including decreased hind limb skeletal muscle mass, muscle fiber atrophy, and reduced cross-sectional area. This successfully simulated clinical features of sarcopenia similar to those seen in humans.

[0351] During the development of disuse-induced muscle atrophy, different muscles experience different rates of muscle fiber loss. The soleus muscle experiences greater loss and greater extent than the gastrocnemius muscle. This study also observed that after spontaneous recovery from hindlimb suspension, the gastrocnemius muscle histomorphology showed no significant lesions and essentially returned to normal compared to the blank control group, while the soleus muscle histomorphology still showed visible muscle fiber atrophy. Measurement of the cross-sectional area of ​​the soleus muscle fibers in each group of mice revealed that the cross-sectional area of ​​the soleus muscle fibers in all TCM-073 dose groups was greater than that in the model control group, with the highest TCM-073 dose (6.00g crude drug / kg) showing the greatest improvement. Grip strength was also tested on the mice. Results showed that during both hindlimb suspension and spontaneous recovery, the high-dose TCM-073 group (6.00g crude drug / kg) showed significantly greater hindlimb grip strength than the model control group, demonstrating that TCM-073 can promote the recovery of grip strength in mice with muscle atrophy. The magnitude of muscle strength is related to the cross-sectional area and number of muscle fibers. Therefore, the improvement of the hind limb grip strength of mice with disuse muscular atrophy by TCM-073 may be related to this.

[0352] Capillaries play a key role in delivering oxygen and nutrients to skeletal muscle. The capillary network in skeletal muscle changes in response to physiological or pathological conditions. Long-term inactivity not only leads to a decrease in skeletal muscle fibers but also to the degeneration of skeletal muscle capillaries. The saphenous vein is a prominent blood vessel on the surface of the gastrocnemius muscle that collects blood from the superficial calf venous layer, which includes the gastrocnemius, soleus, and plantaris muscles. This study used a laser Doppler imager to monitor blood flow in the saphenous vein, demonstrating that a high dose of TCM-073 (6.00g crude drug / kg) significantly increased blood supply to the calf skeletal muscle of mice with disuse-induced muscular atrophy.

[0353] Therefore, the pharmaceutical composition of the present invention may play a certain protective and therapeutic role in the decreased muscle strength and muscle atrophy caused by sarcopenia by increasing the blood supply to muscle tissue and increasing the fullness of muscle fibers.

[0354] 6. Other explanations

[0355] To ensure that the experimental mice were within the same age range, the actual weight range of female mice upon receipt was 17-19 g. In addition, due to the slow growth of female mice, their weight at the beginning of dosing did not reach the expected weight range of the protocol. Considering that this weight would not have a significant impact on the results of this experiment, the experiment was carried out normally as planned.

[0356] 7. Recordkeeping

[0357] In addition to data collected directly by computers or automated instruments, all other data generated during the actual research process must be recorded on spreadsheets or charts and organized and bound at all times. All data records must be signed by the operator and verifier, and the date of recording must be noted. When the final report is submitted, all original data, documents, and other relevant materials must be handed over to the data archive for safekeeping.

[0358] 7.1 Depository: New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd.

[0359] 7.2 Address: No. 238 Tianshan Street, Shijiazhuang City

[0360] Experimental Example 4: Study on the mechanism of the therapeutic effect of the pharmaceutical composition of the present invention on amyotrophic lateral sclerosis SOD1-G93A transgenic mice

[0361] Purpose of the trial

[0362] The mechanism of the therapeutic effect of the pharmaceutical composition of the present invention on amyotrophic lateral sclerosis was explored using SOD1-G93A transgenic mice.

[0363] 1 Experimental Materials

[0364] 1.1 Test sample: The pharmaceutical composition of the present invention, prepared as in Test Example 2, abbreviated as TMC-073.

[0365] 1.2 Positive drugs and main reagents

[0366] 1.2.1 Positive Drug: Riluzole (trade name: Riluzole), 50 mg / tablet, is used to treat amyotrophic lateral sclerosis (ALS), and can prolong patient survival and / or delay tracheotomy. Adults are prescribed 50 mg (1 tablet) orally twice daily. Produced by Sanofi Winthrop Industrie and packaged by Sanofi (Beijing) Pharmaceutical Co., Ltd., batch number: 0KP4C, expiration date: May 2023.

[0367] 1.2.2 Main reagents: Sterile water for injection, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number 2007023202, valid until July 1, 2022.

[0368] 1.3 Experimental System

[0369] 1.3.1 Animal strain: B6SJL-Tg(SOD1-G93A)1Gur / J (Stock Number: 002726) was provided by The Jackson Laboratory, USA. Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. maintained and bred the strain, and identified the offspring SOD1-G93A transgenic mice. Mice from the same littermates that did not carry the human mutant SOD1 gene served as negative controls.

[0370] 1.3.2 Animal level: SPF level.

[0371] 1.3.3 Animal gender and quantity: 80 B6SJL-Tg(SOD1-G93A)1Gur / J mice; 20 mice of the same litter without the human mutant SOD1 gene, both male and female were used.

[0372] 1.3.4 Animal age: 7 - 8 weeks old.

[0373] 1.3.5 Animal weight: At the beginning of drug administration, the weight of male animals was 23.9 g - 31.3 g, and that of female animals was 17.7 g - 25.4 g.

[0374] 1.3.6 Animal source: Purchased from The Jackson Laboratory in the United States and reproduced by Jiangsu Jicui Yakang Biotech Co., Ltd.

[0375] 1.3.7 Animal certificate number, issuing unit, and receiving date: Certificate number 320727210100609023, license number SCXK(Su)2018 - 0008, issuing unit Jiangsu Jicui Yakang Biotech Co., Ltd., receiving date October 13, 2021.

[0376] 1.3.8 Rearing conditions: Mice were caged and reared in the New Drug Evaluation Center of Hebei Institute of Integrated Traditional Chinese and Western Medicine. License number: SYXK(He)2020 - 006. Lighting was 12 hours per day, temperature was 20 - 26 °C, and relative humidity was 40 - 70%.

[0377] 1.3.9 Quarantine process: New animals were adaptively reared for at least 5 days. During this period, it was observed whether the animals drank water and ate normally, had good health conditions, and showed no signs of disease or death.

[0378] 1.3.10 Feed: SPF maintenance feed for mice and rats, provided by Beijing Keao Xieli Feed Co., Ltd. Feed production license number: Jing Si Zheng (2018) 06073, experimental animal production license number: SCXK(Jing)2019 - 0003. Product batch number: 21073213, expiration date: January 3, 2022

[0379] 1.3.11 Drinking water: Clean water prepared by the ROB - B experimental animal drinking water system was used. The drinking water bottles were filled for free drinking. The drinking water was fully tested once a year, mainly including microbial indicators, toxicological indicators, sensory properties, and general chemical indicators, and the testing was entrusted to a qualified institution; Microbiological limit testing was carried out once every 2 months, entrusted to the Quality Inspection Section of Shijiazhuang Yiling Pharmaceutical Co., Ltd. The test reports were kept by the animal room and archived annually.

[0380] 1.3.12 Bedding: Ordinary corncob bedding, sterilized at high temperature and high pressure, provided by Beijing Keao Xieli Feed Co., Ltd., Laboratory Animal Production License No.: SCXK(Beijing)2020-0010. Batch No.: 21099811.

[0381] 1.3.13 Identification: Mouse ear tags are used for labeling. Ear tag manufacturer: Chuangbo Global (Beijing) Biotechnology Co., Ltd.

[0382] 2 Test methods

[0383] 2.1 Basis of experimental design

[0384] 2.1.1 Adoption of standards: Determined by the "Classification and Application Material Requirements for Registration of Traditional Chinese Medicines" and "Good Manufacturing Practice for Non-clinical Drug Research" issued by the State Food and Drug Administration and relevant literature.

[0385] 2.1.2 Experimental System Selection: Transgenic mice carrying a human superoxide dismutase (SOD) gene mutation (B6SJL-Tg(SOD1-G93A)1Gur / J) are currently considered the most commonly used and best-performing animal model for amyotrophic lateral sclerosis (ALS). While the pathogenesis of ALS remains unclear, with 90% of cases occurring sporadically and 10% occurring in familial forms (FALS), the discovery by Rosen in 1993 of a missense mutation in the copper-zinc superoxide dismutase (SOD1) gene was a major milestone in ALS research. The discovery of the SOD1 mutation in FALS led to the subsequent development of animal models for ALS. Currently, the SOD1-G93A transgenic mouse for ALS is widely used in both clinical and basic research. This transgenic mouse carries a high copy number of the human SOD1 mutation. Homozygous mice exhibit a reduction in spinal motor neurons and gradually develop neuromuscular dysfunction, including tail-hanging limb tremors and hemiplegia of one or more limbs, around three months after birth. With a lifespan of approximately four to five months, it mimics the chronic progressive course of ALS and some of its pathological and biochemical manifestations.

[0386] 2.2 Dosage and Grouping

[0387] Based on literature and previous experience, combined with behavioral testing results, ALS mice were randomly divided by body weight and began dosing and observation after showing significant changes compared to negative control mice. Specifically, they were divided into: model control group (M), positive drug control group (riluzole 0.02g / kg, R), TCM-073 low-dose (073-L), medium-dose (073-M), and high-dose (073-H) groups, with 16 animals per group. A negative control group (C, 20 animals total) consisted of wild-type littermate mice that did not carry the human mutant SOD1 gene. Specific experimental groups and doses are shown in Appendix 14.

[0388] Appendix 14: Trial Grouping and Dosage Setting

[0389] 2.3 Administration

[0390] The drug is administered by oral gavage, which is consistent with the clinically recommended oral route.

[0391] 2.4 Preparation and storage of test samples

[0392] The clinical dose of riluzole is approximately 0.002g / kg based on a body weight of 60kg. The dose administered to mice is equivalent to 10 times the clinical dose, approximately 0.02g / kg, with a dosing volume of 10ml / kg and a dosing concentration of 0.02g / 10ml. The drug is prepared by grinding the powder from the tablets. Each tablet contains 50mg of riluzole. For preparation, one tablet is taken and ground to 25ml with sterile water for injection. The amount can be adjusted based on the number of animals.

[0393] The proposed clinical dose of TCM-073 is 18g of crude drug per day. This trial included three doses: low, medium, and high: 1.5, 3.0, and 6.0g of crude drug per kg (equivalent to 5, 10, and 20 times the clinical dosage, respectively). See Appendix 14 for details. Using sterile water for injection as the solvent, the high, medium, and low doses were prepared, and appropriate amounts of drug were weighed and ground.

[0394] 2.5 Administration of test products

[0395] The corresponding drug solution was drawn up with a syringe for oral administration, with a dosage of 10 ml / kg. The model control group and the negative control group were given the corresponding volume of sterile water for injection; the dosage of each animal was adjusted weekly according to the most recent body weight.

[0396] 2.6 Determination of model mice

[0397] Based on previous experimental experience, combined with literature research and the disease characteristics of this strain of mice, drug administration began when the mice were 70 days old.

[0398] 2.7 Observation indicators, time and content

[0399] After 30 days of administration, electrophysiological indicators were tested and samples were collected for subsequent testing.

[0400] 2.7.1 Electrophysiological indicators

[0401] After anesthesia, the animals were placed in a prone position, and recording electrodes were inserted into the gastrocnemius muscle belly of the hind limb. A stimulator was connected to stimulate the lumbar paraspinal sciatic nerve. Stimulation was given, and the peak value, amplitude, and selected time course of the compound muscle action potential were observed and recorded using a Powerlab physiological recorder.

[0402] 2.7.2 Serum malondialdehyde (MDA) index

[0403] Blood was collected from the mouse eyeballs and allowed to stand until the supernatant settled. The blood was then centrifuged at 3000 rpm for 10 minutes at 4°C to separate the serum, which was then aliquoted into EP tubes and stored in an ultra-low temperature freezer (-80°C) for measurement of MDA content. The inhibition rate of oxidative damage by each drug group was calculated based on the MDA content using the following formula:

[0404] 2.7.3 Morphological observation indicators

[0405] The gastrocnemius muscle and sciatic nerve were observed by HE staining; the morphology and number of spinal motor neurons were observed by toluidine blue staining.

[0406] 2.7.4 Immunofluorescence

[0407] Depending on the amount of spinal cord and muscle tissue collected, neuromuscular junctions, stem cell regeneration, microglial senescence, and myelin and neurite damage and repair were assessed. For example, co-staining of the muscle presynaptic marker (synaptotagmin-2) with the acetylcholine receptor (nAChR) marker (α-BTX) was performed; co-staining of the satellite cell marker (Pax7) with laminin 2α was performed; the myelin marker myelin basic protein (MBP) was also performed; and the neurite marker beta-III tubulin (also known as Tuj-1) was also performed.

[0408] 2.7.5 Molecular Biology Indicators

[0409] Based on the amount of spinal cord tissue collected from mice, molecular biological indicators related to myelin and synapses were tested, such as myelin basic protein (MBP) and synaptic vesicle-associated protein (SV2A).

[0410] 2.8 Notification to relevant staff

[0411] Notify the animal room when purchasing animals, and notify the pathology room for treatment when animals show abnormal conditions.

[0412] 2.9 Statistical Methods

[0413] The experimental data are expressed as mean ± standard deviation SPSS 25.0 statistical software was used for statistical analysis. Behavioral scores and tissue detection indicators were first tested for normality. For data that conformed to the normal distribution, one-way analysis of variance was used for mean comparison. If the variances were equal, the least significant difference method was used for pairwise comparison. If the variances were unequal, Dunnett's T3 test was used. If they did not conform to the normal distribution, non-parametric tests were used for statistical analysis.

[0414] 2.10 Instrument System

[0415] JJ2000 electronic balance, Changshu Shuangjie Testing Instrument Factory.

[0416] BT224S Sartorius precision analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0417] NVT2201ZH electronic balance, Ohaus Instruments (Shanghai) Co., Ltd.

[0418] Mettler ME104E analytical balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0419] DT2000 electronic balance, Changshu Shuangjie Testing Instrument Factory.

[0420] PowerLab physiological recorder, Ade Instrument International Trading (Shanghai) Co., Ltd.

[0421] Inverted fluorescence confocal microscope, Zeiss.

[0422] Mettler electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0423] SYG-4 digital display constant temperature water bath, Changzhou Langyue Instrument Manufacturing Co., Ltd.

[0424] Gene Speed ​​1580R high-speed refrigerated centrifuge, Gene Co., Ltd.

[0425] TECAN Infinite M200Pro microplate reader, TECAN, Switzerland.

[0426] WS-350P microplate fast shaker, Wiggens Labortechnik GmbH.

[0427] SE260 vertical electrophoresis tank, Ge Healthcare UK Ltd

[0428] EPS301 electrophoresis instrument, Ge Healthcare Bio-Sciences AB

[0429] eBlot L1 fast wet transfer instrument, GenScript Biotech Co., Ltd.

[0430] WD-9405DN digital display horizontal shaker, Beijing Liuyi Biotechnology Co., Ltd.

[0431] Odyssey clx near-infrared dual-color laser imaging system, LI-COR.

[0432] Multi-sample tissue grinder, Shanghai Jingxin Industrial Development Co., Ltd.

[0433] ZWYR-240 constant temperature culture oscillator, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.

[0434] CHB-202 constant temperature metal bath, Hangzhou Biori Technology Co., Ltd.

[0435] BPN-CH CO2 incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.

[0436] 3 Results

[0437] 3.1 Effects on muscle action potential

[0438] As shown in Appendix Tables 15, 16, and 17, the model control group had significantly lower action potential peaks (P<0.001), significantly lower amplitudes (P<0.001), and significantly prolonged durations of the selected area (P<0.001) compared with the negative control group. The action potential peaks of the positive drug and each drug-treated group were significantly higher than those of the model group (P<0.05, P<0.01). The action potential amplitudes of the positive drug (P<0.05) and the low-dose (P<0.05) and high-dose (P<0.01) TCM-073 groups were significantly higher than those of the model group. The durations of the selected area were significantly shorter (P<0.05) than those of the positive drug and each drug-treated group (P<0.01).

[0439] Appendix 15 Action potential peak ###P<0.001,*P<0.05,**P<0.01,#negative control group vs. model control group, *medication group vs. model control group

[0440] Appendix 16 Action potential amplitude ###P<0.001,*P<0.05,**P<0.01,#negative control group vs. model control group, *medication group vs. model control group

[0441] Appendix 17 Action potential selection time course ###P<0.001,*P<0.05,**P<0.01,#negative control group vs. model control group, *medication group vs. model control group

[0442] 3.2 Effect on serum malondialdehyde (MDA) content

[0443] After removing the eyeballs of mice and collecting blood, the blood was allowed to stand until the supernatant settled. The blood was then centrifuged at 3000 rpm for 10 minutes at 4°C, and the serum was separated. MDA levels were measured using a kit, and the inhibition rate of each drug group on oxidative damage was calculated based on the MDA levels. The results are shown in Appendix 18. MDA levels were significantly increased in the model control group compared with the negative control group (P < 0.01). The positive drug and all treatment groups significantly reduced MDA levels (P < 0.05, P < 0.01). Serum malondialdehyde levels were significantly reduced in the medium (P < 0.05) and high-dose (P < 0.01) TCM-073 groups compared with the positive drug group.

[0444] Appendix 18 Serum malondialdehyde (MDA) content ##P<0.01,*P<0.05,**P<0.01,&P<0.05,&&P<0.01,#Negative control group vs. model control group,*Drug treatment group vs. model control group,&Positive drug control group vs. Drug treatment group

[0445] 3.3 Impact on morphological observation indicators

[0446] HE staining of the sciatic nerve revealed that the primary lesions in the model control group were axonal degeneration and myelin degeneration, with slightly more severe lesions in males. Compared with the model control group, the lesions in the positive drug control group and the high-dose TCM-073 group were slightly milder, while no significant improvement was observed in the other treatment groups. See Appendix 19 for details.

[0447] Appendix 19 Pathology Report

[0448] in conclusion:

[0449] In summary, pathological examination results from this trial showed that the primary lesions in the model control group were sciatic nerve axonal degeneration accompanied by myelin degeneration, with slightly more severe lesions in males. Compared with the model control group, the lesions in the positive drug control group and the high-dose TCM-073 group were slightly milder, while no significant improvement was observed in the other treatment groups. See Table 20 for details.

[0450] ______________________________________________________________

[0451] Appendix 20: Scoring results of main lesions of individual animals Note 1. Lesion grading: none (-), slight (+), mild (++), moderate (+++), severe (++++). Note 2. Group scores are calculated based on the median of the lesion severity ratings of the animals in the group ("-" represents 0 points, each "+" represents 1 point, and the higher the score, the more severe the lesion).

[0452] 3.4 Effects on the degree of damage and repair of neuromuscular junctions, muscles, myelin sheaths, and neurites

[0453] The results of immunofluorescence detection of gastrocnemius muscle presynaptic markers (synaptotagmin-2) and acetylcholine receptor (nAChR) markers (α-BTX) are shown in Figure 21. Compared with the negative control group, the co-localization degree of synaptotagmin-2 and α-BTX was reduced in the model control group, and the co-localization degree was increased in the positive drug and each drug-treated group.

[0454] The results of immunofluorescence detection of gastrocnemius satellite cell markers (Pax7) and laminin (Laminin 2α) are shown in Figure 22. Compared with the negative control group, the model control group had fewer Pax7-positive cells, while the positive drug and each drug-treated group had an increase in positive cells, and Laminin 2α marked the cell outline.

[0455] The results of immunofluorescence detection of spinal cord myelin marker myelin basic protein (MBP) are shown in Figure 23. The expression of MBP in the model control group was reduced compared with the negative control group, while the expression was increased in the positive drug and each drug-treated group.

[0456] The results of immunofluorescence detection of spinal cord neurite markers (beta-III Tubulin also known as Tuj-1) are shown in Figure 24. The expression of Tuj-1 in the model control group was reduced compared with the negative control group, while the expression of Tuj-1 in the positive drug and all drug-treated groups was increased. 3.5 Effects on molecular biological indicators related to spinal cord myelin and synapses

[0457] Based on the amount of spinal cord collected from the mice, molecular biological indicators of myelin and synapses were assessed. Results for myelin basic protein (MBP) and synaptic vesicle-associated protein (SV2A) are shown in Appendix Tables 21 and 22. Immunoblotting results showed that MBP expression was significantly lower in the model control group compared with the negative control group (P < 0.05), while MBP levels were significantly increased in the positive drug group and in all treatment groups (P < 0.05). SV2A expression was significantly lower in the model control group compared with the negative control group (P < 0.001), while the high-dose TCM-073 group significantly increased SV2A levels (P < 0.01), reaching levels significantly higher than those in the positive drug group (P < 0.01).

[0458] Appendix 21 Spinal cord MBP content #P<0.05, *P<0.05, #negative control group vs. model control group, *medication group vs. model control group

[0459] Appendix 22 Spinal cord SV2A content ###P<0.001,**P<0.01,&&P<0.01,#Negative control group vs. model control group,*Drug treatment group vs. model control group,& Positive drug control group vs. Drug treatment group

[0460] 4 Conclusion

[0461] The experiment used SOD1-G93A transgenic mice, a classic model of ALS, and began administering drugs at 70 days of age to evaluate the therapeutic effects of different doses of TCM-073. The results showed that each dosage group of the test drug could increase the peak value of muscle action potential and shorten the selected area time course, among which the low and high dose groups (1.5g, 6.0g crude drug / kg) could increase the action potential amplitude. Each administration group could significantly reduce serum malondialdehyde content (P < 0.01). Among them, the medium and high dose groups of TCM-073 (3.0g, 6.0g crude drug / kg) were significantly better than the positive drug (P < 0.05, P < 0.01). HE staining of the sciatic nerve showed that compared with the model control group, the positive drug control group and the high dose group of TCM-073 (6.0g crude drug / kg) had slightly lighter lesions, while the lesions of the other administration groups did not show significant improvement. Immunofluorescence analysis revealed that both the positive drug and all drug-treated groups increased neuromuscular junction colocalization, the number of positive satellite cells in the gastrocnemius muscle, and the expression of myelin and neurite markers. Western blotting revealed that both the positive drug and all drug-treated groups significantly increased spinal cord MBP levels (P < 0.05). The high-dose TCM-073 group (6.0 g crude drug / kg) significantly increased SV2A levels (P < 0.01), significantly exceeding those in the positive drug group (P < 0.01).

[0462] 5. Recordkeeping

[0463] In addition to data collected directly by computers or automated instruments, all other data generated during the actual research process must be recorded on spreadsheets or charts and organized and bound at all times. All data records must be signed by the operator and verifier, and the date of recording must be noted. When the final report is submitted, all original data, documents, and other relevant materials must be handed over to the data archive for safekeeping.

[0464] 5.1 Depository: New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd.

[0465] 5.2 Address: No. 238 Tianshan Street, Shijiazhuang City

[0466] Experimental Example 5: Study on the mechanism of therapeutic effect of the pharmaceutical composition of the present invention on CPZ-induced demyelination model mice

[0467] Purpose of the trial

[0468] The therapeutic effect of the pharmaceutical composition of the present invention on central nervous system demyelination was observed using CPZ-induced demyelination model mice.

[0469] 1 Experimental materials

[0470] 1.1 Test samples: The pharmaceutical composition of the present invention, prepared as in Test Example 2, abbreviated as TMC-073.

[0471] 1.2 Main reagents

[0472] Sterile water for injection, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number 2009053201, expiration date September 4, 2022.

[0473] 1.3 Experimental system

[0474] 1.3.1 Animal strain: C57BL / 6J.

[0475] 1.3.2 Animal level: SPF level.

[0476] 1.3.3 Animal sex and quantity: Male, 60 (58 selected for the experiment).

[0477] 1.3.4 Animal age: 42 - 48 days old.

[0478] 1.3.5 Animal weight at the start of dosing: 24.1 g - 31.7 g.

[0479] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0480] 1.3.7 Animal certificate number, issuing unit, and receiving date: Certificate number 110011220103270315, license number SCXK(Beijing) 2021 - 0006, issuing unit Beijing Vital River Laboratory Animal Technology Co., Ltd., receiving date March 30, 2022.

[0481] 1.3.8 Rearing conditions: Mice are caged and reared in the New Drug Evaluation Center of Hebei Academy of Integrative Medicine and Western Pharmacy, license number: SYXK(Hebei) 2020 - 006. Lighting is 12 hours per day, temperature is 20 - 26 °C, and relative humidity is 40 - 70%.

[0482] 1.3.9 Quarantine process: New animals are adaptively reared for at least 5 days, during which it is observed whether the animals drink water and eat normally, have good health, and show no signs of disease or death.

[0483] 1.3.10 Feed: SPF rat maintenance feed, provided by Sibeifu (Beijing) Biotechnology Co., Ltd., Laboratory Animal Production License No.: SCXK (Beijing) 2019-0010. Product batch number: 0112SH01140404C, valid until July 13, 2022. 0.2% cuprizone-doped feed, provided by Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd., Laboratory Animal Production License No.: SCXK (Beijing) 2018-0006, production date: 20220314, valid until June 13, 2022.

[0484] 1.3.11 Drinking Water: Drink clean water prepared by the ROB-B experimental animal drinking water system, filled in drinking water bottles for free drinking. Drinking water is fully tested once a year, mainly including microbiological indicators, toxicological indicators, sensory properties and general chemical indicators, and the testing is entrusted to a qualified institution; microbial limit testing is conducted every two months, entrusted to the Quality Inspection Department of Shijiazhuang Yiling Pharmaceutical Co., Ltd., and the test report is kept by the animal room and archived annually.

[0485] 1.3.12 Bedding: Ordinary corncob bedding, sterilized at high temperature and high pressure, provided by Beijing Keao Xieli Feed Co., Ltd., Laboratory Animal Production License No.: SCXK(Beijing)2020-0010. Batch No.: 22019811, 22049811.

[0486] 1.3.13 Identification: Mouse ear tags are used for labeling. Ear tag manufacturer: Chuangbo Global (Beijing) Biotechnology Co., Ltd.

[0487] 2 Test methods

[0488] 2.1 Basis of experimental design

[0489] 2.1.1 Adoption of standards: Determined by the "Classification and Application Documents Requirements for Registration of Traditional Chinese Medicines" and "Good Manufacturing Practice for Non-clinical Drug Research" issued by the State Food and Drug Administration and relevant literature.

[0490] 2.1.2 Experimental System Selection: Oral administration of cuprizone (CPZ) induces oligodendrocyte apoptosis within a few days, followed by apoptosis of innate immune cells in the brain, namely astrocytes and microglia, ultimately leading to demyelination in distinct white and gray matter regions. Therefore, this model reflects several key features of the ongoing demyelinating process. The CPZ model can be used to develop and study drugs and mechanisms related to demyelinating pathologies. Estrogen has been shown to counteract CPZ-induced demyelination, so only male animals were used in this study. Acute demyelination is induced in 6-8 week-old mice by oral administration of a diet containing 0.2% CPZ for 6 weeks. Following a 2-week switch to a normal diet, the mice undergo spontaneous endogenous remyelination. During this period, administering the drug under investigation can be used to assess its ability to accelerate the ongoing remyelination process.

[0491] 2.2 Dosage and Grouping

[0492] After 6 weeks of feeding with 0.2% CPZ and demonstrating a significant difference in Weaver's 15-point score compared to vehicle control mice, mice were randomly divided into groups based on score and body weight and began dosing observation. The groups included: vehicle control (C), model control (M), TCM-073 low-dose (073-L), medium-dose (073-M), and high-dose (073-H) groups. The vehicle control group consisted of 10 mice, and the remaining groups consisted of 12 mice each. Specific experimental groupings and doses are shown in Appendix 23.

[0493] Appendix 23 Trial Grouping and Dosage Setting Table

[0494] 2.3 Administration

[0495] The drug is administered by oral gavage, which is consistent with the clinically recommended oral route.

[0496] 2.4 Preparation and storage of test samples

[0497] The proposed clinical dose of TCM-073 is 18g of crude drug per day. This trial employed three doses: low, medium, and high: 1.5, 3.0, and 6.0g of crude drug per kg (equivalent to 5, 10, and 20 times the clinical dosage, respectively). See Appendix 23 for details. Using sterile water for injection as the solvent, the high, medium, and low doses were prepared, and appropriate amounts of drug were weighed and ground.

[0498] 2.5 Administration of test products

[0499] The corresponding drug solution was drawn out with a syringe for oral administration at a volume of 10 ml / kg. The model control group and the solvent control group were given corresponding volumes of sterile water for injection.

[0500] 2.6 Determination of model mice

[0501] 2.6.1 Weaver's 15-point scoring system: Tail activity is divided into three levels, and limb activity is divided into four levels. The cumulative score is used to determine the score (see Appendix 24). If death occurs, the total score is 15. Literature research suggests that mice fed a 0.2% CPZ diet for 6 weeks develop acute demyelination symptoms.

[0502] Appendix 24 Weaver's 15-point scoring system Note: The paralysis of the tail and each limb is evaluated one by one, and the total score is obtained by adding up.

[0503] 2.7 Observation indicators, time and content

[0504] During the drug administration phase, neurological function was evaluated weekly (Weaver's 15-point scoring method), and rotarod and open field tests were performed 14 days after drug administration.

[0505] 2.7.1 Rotarod

[0506] A rotarod test was performed to assess each mouse's sensorimotor coordination. The speed was set at 30 rpm for 120 s. The duration of each mouse's time on the rotarod was recorded.

[0507] 2.7.2 Open Field

[0508] The open field test equipment consists of an open field chamber and an automated data acquisition and processing system. A camera is mounted directly above the chamber, capturing the entire open field. First, place the mouse in the center of the chamber, and simultaneously begin recording and timing. The total distance covered by the mouse's spontaneous movements over a 5-minute period is recorded.

[0509] 2.8 Notification to relevant staff

[0510] Notify the animal room when purchasing animals, and notify the pathology room for treatment when animals show abnormal conditions.

[0511] 2.9 Statistical Methods

[0512] The experimental data are expressed as mean ± standard deviation SPSS 25.0 statistical software was used for statistical analysis. Behavioral scores and tissue detection indicators were first tested for normality. For data that conformed to the normal distribution, one-way analysis of variance was used for mean comparison. If the variances were equal, the least significant difference method was used for pairwise comparison. If the variances were unequal, Dunnett's T3 test was used. If they did not conform to the normal distribution, non-parametric tests were used for statistical analysis.

[0513] 2.10 Instrument System

[0514] Sartorius BSA2201 electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0515] BT224S Sartorius precision analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0516] IITC Rotarod Fatigue Apparatus for Rats and Mice, IITC Biological Sciences.

[0517] ANY-maze small animal behavior instrument, Jiangsu Saiangsi Biotechnology Co., Ltd.

[0518] 3 Results

[0519] 3.1 Effects on nervous system function

[0520] As shown in Appendix 25, the model control group showed significantly higher neurological scores compared to the vehicle control group on days 7 and 14 of drug administration (P<0.001). No other drug-administered groups showed a decrease in neurological scores on day 7. On day 14, the TCM-073 medium (P<0.05) and high-dose groups (P<0.01) showed a significant decrease in neurological scores compared to the model group.

[0521] Appendix 25 Neurological score ###P<0.001,*P<0.05,**P<0.01,#Vehicle control group vs. model control group, *Drug-treated group vs. model control group

[0522] 3.2 Effects on Rotarod Duration

[0523] The rotarod test was used to assess each mouse's sensorimotor coordination ability. The speed was set at 30 rpm for 120 seconds. The duration of each mouse's rotarod was recorded. The results are shown in Appendix Table 26. The model control group showed a significantly shorter rotarod duration compared to the vehicle control group (P < 0.001). All drug-treated groups showed a significantly longer rotarod duration (P < 0.05, P < 0.01).

[0524] Table 26 Rotarod Duration ###P<0.001,*P<0.05,**P<0.01,#Vehicle control group vs. model control group, *Drug-treated group vs. model control group

[0525] 3.3 Impact on open field test

[0526] The mice were placed in the center area of ​​the open field reaction box, and video recording and timing were started at the same time. The total distance, average speed, and number of circling of the mice's spontaneous activities within 5 minutes were recorded. In the demyelination model, myelin loss can lead to abnormal nerve signal conduction in mice, resulting in symptoms of anxiety and irritability such as increased spontaneous activity and increased number of circling. The results are shown in Appendix 27. Compared with the solvent control group, the model control group had a significantly longer activity distance (P < 0.05), a significantly faster average speed (P < 0.05), and a significantly increased circling speed (P < 0.05). All drug-treated groups significantly shortened the movement distance, slowed the average speed, and reduced the number of circling (P < 0.05, P < 0.01).

[0527] Appendix 27 Open field test data ###P<0.001,*P<0.05,**P<0.01,#Vehicle control group vs. model control group, *Drug-treated group vs. model control group

[0528] 4 Conclusion

[0529] The experiment used CPZ to induce a demyelination model in mice to evaluate the therapeutic effects of different doses of the pharmaceutical composition of the present invention. The results showed that the medium and high dose groups of the test drug significantly reduced the neurological score (P<0.05, P<0.01). In behavioral indicators, all the groups treated with the test drug significantly prolonged the duration of the rotarod; shortened the open field activity distance, reduced the average speed and reduced the number of circles (P<0.05, P<0.01). This suggests that the pharmaceutical composition of the present invention has the effect of protecting against damage to nerve function caused by demyelination.

[0530] 5. Recordkeeping

[0531] In addition to data collected directly by computers or automated instruments, all other data generated during the actual research process must be recorded on spreadsheets or charts and organized and bound at all times. All data records must be signed by the operator and verifier, and the date of recording must be noted. When the final report is submitted, all original data, documents, and other relevant materials must be handed over to the data archive for safekeeping.

[0532] 5.1 Depository: New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd.

[0533] 5.2 Address: No. 238 Tianshan Street, Shijiazhuang City

[0534] 5.3 Postal Code: 050035

[0535] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating amyotrophic lateral sclerosis, characterized in that: The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 3-9 parts of ginseng and 3-9 parts of Cistanche deserticola.

2. The Chinese medicine composition according to claim 1, characterized in that The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 4-6 parts of ginseng and 4-6 parts of Cistanche deserticola.

3. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is composed of the following raw materials in parts by weight: 3 parts of ginseng and 9 parts of Cistanche deserticola.

4. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is composed of the following raw materials in parts by weight: 6 parts of ginseng and 6 parts of Cistanche deserticola.

5. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is composed of the following raw materials in parts by weight: 9 parts of ginseng and 3 parts of Cistanche deserticola.

6. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is composed of the following raw materials in parts by weight: 7 parts of ginseng and 4 parts of Cistanche deserticola.

7. The method for preparing the Chinese medicine composition according to any one of claims 1 to 6, characterized in that: The active ingredients of the Chinese medicine composition are prepared by the following steps: Ginseng and Cistanche deserticola are weighed according to the prescribed amount, boiled with water, filtered, and the filtrate is concentrated under reduced pressure to obtain an extract, which is the active ingredient of the traditional Chinese medicine composition.

8. Use of the Chinese medicine composition according to any one of claims 1 to 6 or the Chinese medicine composition prepared by the preparation method according to claim 7 in the preparation of a medicament for treating amyotrophic lateral sclerosis.

9. The use according to claim 8, characterized in that: The amyotrophic lateral sclerosis is manifested by one or more of decreased muscle strength, muscle atrophy, muscle twitching, muscle stiffness or weakness, slurred speech, difficulty swallowing, shortness of breath, soreness of waist and knees, fatigue and laziness, or chills and cold limbs.

10. Use of the Chinese medicine composition according to any one of claims 1 to 6 in the preparation of drugs for delaying the paralysis time of amyotrophic lateral sclerosis, improving damaged nerve function, alleviating sciatic nerve lesions, and increasing the co-localization degree of neuromuscular junctions.

11. Use of the Chinese medicine composition according to any one of claims 1 to 6 in preparing a drug for prolonging the hanging time of mice, increasing the peak value of muscle action potential and shortening the selection time.

12. Use of the Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a drug for improving symptoms of demyelination in the central nervous system, improving muscle strength and muscle fiber fullness, and increasing blood supply to muscle tissue.

13. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a drug for treating sarcopenia.

14. The Chinese medicine composition as described in any one of claims 1 to 6 is used in the preparation of improving neuroelectrophysiology, reducing serum malondialdehyde content, promoting axonal growth and remyelination, protecting nerve function, improving muscle presynaptic markers and acetylcholine receptor marker colocalization degree, improving gastrocnemius satellite cell positive number and myelin marker (MBP), improving the integrity of neuromuscular junction structure, increasing the expression of neurite marker (Tuj-1) and synaptic vesicle protein (SV2A), promoting remyelination, improving behavioral changes caused by demyelination or improving motor neuron signal transmission to muscle, and delaying the application of disease progression drugs for amyotrophic lateral sclerosis.

15. A medicine comprising the Chinese medicine composition according to any one of claims 1 to 6 or the Chinese medicine composition prepared by the preparation method according to claim 7.

16. The drug according to claim 15, characterized in that The dosage form is capsule, tablet, granule, injection, pill, powder, spray or oral solution.

17. The drug according to claim 16, characterized in that The granules are prepared by the following steps: (1) Weigh ginseng and Cistanche deserticola according to the prescribed amount, add water and boil until complete, filter the water extract, and concentrate the filtrate to extract for later use; (2) taking the extract, spray drying it with a spray dryer, and collecting the spray powder for later use; (3) Add appropriate amount of auxiliary materials to the spray powder obtained in step (2) and granulate it into granules according to conventional methods.

18. The drug according to claim 16, characterized in that The granules are prepared by the following steps: (1) Weigh ginseng and Cistanche deserticola according to the prescribed amount, add water and boil three times, add 10 times the amount of water for the first time, and add 8 times the amount of water for the second and third times, boil for 1.5 hours each time, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.05-1.10 (60°C) for later use; (2) taking the extract, spray drying it with a spray dryer, and collecting the spray powder for later use; (3) Add appropriate amount of auxiliary materials to the spray powder obtained in step (2) and granulate it into granules according to conventional methods.

19. A method for treating a disease, comprising administering the Chinese medicine composition according to any one of claims 1 to 6, or administering the Chinese medicine composition prepared by the preparation method according to claim 7, or administering the medicine according to any one of claims 15 to 18.

20. The method of claim 19, wherein: The treatment includes at least one of the following: Delay the paralysis of amyotrophic lateral sclerosis, improve damaged nerve function, reduce sciatic nerve lesions, and increase the co-localization of neuromuscular junctions; Prolonged the hanging time of mice, increased the peak value of muscle action potential and shortened the selection time course; Improve the symptoms of demyelination in the central nervous system, increase muscle strength and muscle fiber fullness, and increase blood supply to muscle tissue; Improve neuroelectrophysiology, reduce serum malondialdehyde content, promote axon growth and myelin regeneration, protect nerve function, increase the co-localization of muscle presynaptic markers and acetylcholine receptor markers, increase the number of positive gastrocnemius satellite cells and myelin marker (MBP), improve the integrity of neuromuscular junction structure, increase the expression of neurite markers (Tuj-1) and synaptic vesicle protein (SV2A), promote myelin regeneration, improve behavioral changes caused by demyelination or improve signal transmission from motor neurons to muscles, and delay the progression of amyotrophic lateral sclerosis.