Cannabinoid composition and application thereof in preparation of drug for treating neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease

Abstract

Embodiments of the present invention provide a cannabinoid composition and an application thereof in the preparation of a drug for treating neurodegenerative diseases, so as to solve the problem of the invention and other problems of conventional drugs for treating neurodegenerative diseases, such as large side effects and poorer efficacy after long-term administration.

Description

A cannabinoid composition and its use in preparing medicines for treating neurodegenerative diseases such as Parkinson's and Alzheimer's Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a cannabinoid composition and its use in preparing medicines for treating neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Background Art

[0002] Neurodegeneration associated with Parkinson's disease (PD), Alzheimer's disease (AD), and dementia with Lewy bodies (DLB) is a growing health burden in the aging population. Among those over 80, one in two will develop Alzheimer's disease, with a mortality rate second only to heart disease, cancer, and stroke. The prevalence of Parkinson's disease is 1% to 2% among those over 65, and approximately 4% among those over 85.

[0003] Through long-term research, these neurodegenerative diseases are gradually being linked to abnormal dopamine metabolism and a decrease in dopamine-secreting neurons due to loss. The hallmark pathological changes of AD are neuritic plaques outside neurons and fibrillary tangles within neurons. Currently, cholinesterase inhibitors are the primary treatment, but research has revealed that the neurotransmitter dopamine is also involved in the Alzheimer's disease process. Autopsies of Alzheimer's patients have revealed decreased dopamine levels, L-DOPA, and its metabolites in the striatum, amygdala, and substantia nigra.

[0004] The characteristic pathological changes of Parkinson's disease (PD) are the massive degeneration and death of dopaminergic (DA) neurons in the substantia nigra of the midbrain, as well as the formation of Lewy bodies in the cytoplasm of the remaining neurons. Currently, the main treatments for PD include traditional medications and surgical interventions. Medication includes dopamine replacement therapy, anticholinergic agents, dopamine receptor agonists, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and glutamate receptor antagonists. Levodopa is a commonly used medication for Parkinson's disease and is considered the most effective treatment. However, long-term use of many medications, including levodopa, not only leads to decreased efficacy but also causes various complications, such as on-off phenomena, wearing-off, and movement disorders.

[0005] Therefore, we urgently need to find a drug with high efficacy, long-lasting effect and few side effects.

[0006] Disclosure of the invention

[0007] The present invention addresses the problems that existing drugs used to treat neurodegenerative diseases such as Parkinson's disease have significant side effects and poor efficacy after long-term use. It provides a cannabinoid composition and its use in the preparation of drugs for treating neurodegenerative diseases.

[0008] Cannabidiol (CBD) is a non-toxic, high-value-added phenolic substance extracted from cannabis flowers and leaves that can be used in medicines, cosmetics, and health foods. At present, developed countries such as the United States and the United Kingdom have used it as a raw material and developed a variety of special-effect medicines. CBD is a non-addictive component in cannabis that can hinder the effects of THC on the human nervous system and has pharmacological activities such as anti-spasmodic, anti-rheumatoid arthritis, and anti-anxiety. There are also reports on its use in the treatment of Parkinson's disease, but the therapeutic effect of using CBD alone is not ideal. Although there are studies covering compound cannabinoid combinations, most combinations contain the addictive substance THC. Considering that various countries have more or less control measures on THC, we believe that in the long run, it is more important to develop new therapeutic drugs with good efficacy and fewer side effects without using THC.

[0009] Cannabinol (CBN) is a cannabinoid derived from the oxidation and decomposition of THC, which largely reduces its psychoactive properties.

[0010] Cannabigerol (CBG), a precursor to other cannabinoids such as CBD and THC, is highly effective and free of psychotoxicity and other side effects. While there has been extensive research on the use of CBD and THC in the treatment of neurodegenerative diseases, there are relatively few reports on the therapeutic effects of CBG.

[0011] Based on the above, the first object of the present invention is to provide a cannabinoid composition, which comprises cannabidiol and cannabigerol, and the mass ratio of cannabidiol to cannabigerol is 1:1 to 1:10 or 1:0.3 to 1:0.5 or 1:0.5 to 1:0.7 or 1:0.7 to 1:1.

[0012] Preferably, the mass ratio of cannabidiol to cannabigerol is 1:1 to 1:3 or 1:3 to 1:5 or 1:5 to 1:7.

[0013] Preferably, the cannabinoid composition further comprises cannabinol.

[0014] Preferably, the mass ratio of cannabidiol to cannabinol is 1:(0.05-1).

[0015] Preferably, the mass ratio of cannabidiol to cannabinol is 1:(0.1-0.5).

[0016] Preferably, the mass ratio of cannabidiol, cannabigerol and cannabinol is 1:3:0.3.

[0017] Another object of the present invention is to provide the use of the cannabinoid composition in the preparation of a medicament for treating neurodegenerative diseases;

[0018] Preferably, the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, and Lewy body dementia;

[0019] The cannabinoid composition is used to promote the development and maturation of dopamine nerve cells, prevent dopamine nerve cell shedding, and improve the viability of dopamine nerve cells;

[0020] The cannabinoid composition is used to enhance the ability of dopamine nerve cells to secrete dopamine.

[0021] Another object of the present invention is to provide a pharmaceutical composition for treating neurodegenerative diseases, wherein the pharmaceutical composition comprises: the cannabinoid composition and a pharmaceutically acceptable carrier.

[0022] The dosage forms of the pharmaceutical composition include oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, and atomizers.

[0023] Another object of the present invention is to provide a drug kit for treating neurodegenerative diseases, wherein the drug kit comprises: the cannabinoid composition.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses an experimental method of inducing fibroblasts to differentiate into dopaminergic neuron cell models, detects the cell morphology, quantity and relative expression levels of DA-related factors after induced differentiation and culture of fibroblasts, screens the effects of different common cannabinoids and their combinations on the induction and differentiation of fibroblasts into dopaminergic neurons, and finds that the combination of CBD + CBG has the best effect in improving the viability and dopamine secretion ability of dopaminergic neuron cells, and can be used to prepare drugs for the treatment of neurodegenerative diseases such as Parkinson's disease. Further experiments were designed to combine CBD and CBG in different ratios, and it was found that when the mass ratio of CBD and CBG was 1:1 to 1:5, the experimental effect was better and safer, among which the optimal mass ratio was 1:3.

[0026] (2) CBD+CBG is the most effective combination because it does not contain the addictive substance THC, thus greatly reducing the side effects of drug use. There is no need to worry about drug abuse, which removes a huge obstacle at the application level.

[0027] (3) The CBD+CBG combination provided by the present invention can significantly increase the relative expression levels of Pitx3 and TH-2, that is, it can improve the viability of dopaminergic neuronal cells and the ability to secrete dopamine. This mechanism of action is superior to existing commonly used Parkinson's treatment options (such as oral levodopa). In the long run, it will not produce the long-term use defects similar to levodopa, and will not make the effect worse. At the same time, due to the different mechanisms of action, it is theoretically possible to use it together with drugs such as levodopa to achieve better therapeutic effects, or greatly delay the shortcomings of long-term use of levodopa.

[0028] (4) Based on the combination of CBD+CBG, the present invention further designed experiments on different ratios of CBD, CBG and CBN. The results showed that the addition of CBN further enhanced the effect of the cannabinoid composition in activating the activity of dopamine nerve cells. When the CBD:CBG:CBN (mass ratio) was 1:3:0.3, the effect of activating the activity of dopamine nerve cells was optimal.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a cell morphology image of HDF38 fibroblasts observed under a microscope on the 13th day of induced differentiation culture in Example 1 of the present invention.

[0031] Figure 2 shows the relative expression levels of DA-related factors in each group of cells in Example 1 of the present invention.

[0032] A is the expression detection result of Nurr1;

[0033] B is the expression detection result of TH-2;

[0034] C is the expression detection result of Pitx3.

[0035] In Figures 1 and 2: Cont represents the non-induced differentiation group; 6CD represents the induced differentiation control group; 6CD+d represents the induced differentiation + CBD group; 6CD+g represents the induced differentiation + CBG group; 6CD+t represents the induced differentiation + THC group; 6CD+dg represents the induced differentiation + CBD + CBG group; 6CD+dt represents the induced differentiation + CBD + THC group.

[0036] FIG3 is a microscopic view of the cell morphology of HDF38 fibroblasts induced and differentiated in Example 2 of the present invention; wherein:

[0037] A is the cell morphology on the 4th day of induction culture;

[0038] B is the cell morphology on the 7th day of induction culture;

[0039] C is the cell morphology on the 13th day of induction culture.

[0040] Figure 4 shows the relative expression levels of DA-related factors in each group of cells in Example 2 of the present invention.

[0041] A is the expression detection result of Nurr1;

[0042] B is the expression detection result of TH-2;

[0043] C is the expression detection result of Pitx3.

[0044] In Figures 3 and 4: Cont represents the non-induced differentiation group; 6CD represents the induced differentiation control group; 6CD+dg0.1 represents the addition of CBD and CBG at a mass ratio of 1:0.1; 6CD+dg0.3 represents the addition of CBD and CBG at a mass ratio of 1:0.3; 6CD+dg0.5 represents the addition of CBD and CBG at a mass ratio of 1:0.5; 6CD+dg0.7 represents the addition of CBD and CBG at a mass ratio of 1:0.7; 6CD+dg1 represents the addition of CBD and CBG at a mass ratio of 1:1; 6CD+dg3 represents the addition of CBD and CBG at a mass ratio of 1:3; 6CD+dg5 represents the addition of CBD and CBG at a mass ratio of 1:5; 6CD+dg7 represents the addition of CBD and CBG at a mass ratio of 1:7; 6CD+dg10 represents the addition of CBD and CBG at a mass ratio of 1:10; 6CD+dt represents the induced differentiation + CBD + THC group.

[0045] Figure 5 shows the relative expression levels of DA-related factors in each group of cells in Example 3 of the present invention.

[0046] A is the expression detection result of Nurr1;

[0047] B is the expression detection result of TH-2;

[0048] C is the expression detection result of Pitx3.

[0049] In Figure 5: Cont represents the non-induced differentiation group; 6CD represents the induced differentiation control group; 6CD+dg1 represents the addition of CBD and CBG at a mass ratio of 1:1; 6CD+dg1+n0.1 represents the addition of CBD, CBG and CBN at a mass ratio of 1:1:0.1; 6CD+dg1+n0.3 represents the addition of CBD, CBG and CBN at a mass ratio of 1:1:0.3; 6CD+dg1+n0.5 represents the addition of CBD, CBG and CBN at a mass ratio of 1:1:0. .5 of CBD, CBG and CBN; 6CD+dg3 means adding CBD and CBG in a mass ratio of 1:3; 6CD+dg3+n0.1 means adding CBD, CBG and CBN in a mass ratio of 1:3:0.1; 6CD+dg3+n0.3 means adding CBD, CBG and CBN in a mass ratio of 1:3:0.3; 6CD+dg3+n0.5 means adding CBD, CBG and CBN in a mass ratio of 1:3:0.5.

[0050] Best Mode for Carrying Out the Invention

[0051] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0052] The present invention discloses a cannabinoid composition, which comprises cannabidiol and cannabigerol, wherein the mass ratio of cannabidiol to cannabigerol is 1:1 to 1:10; preferably, the mass ratio of cannabidiol to cannabigerol is 1:1 to 1:3 or 1:3 to 1:5 or 1:5 to 1:7; more preferably, the mass ratio of cannabidiol to cannabigerol is 1:3.

[0053] The cannabinoid composition also contains cannabinol, wherein the mass ratio of cannabidiol to cannabinol is 1:(0.05-1); preferably, the mass ratio of cannabidiol to cannabinol is 1:(0.1-0.5); more preferably, the mass ratio of cannabidiol, cannabigerol and cannabinol is 1:3:0.3.

[0054] Experimental studies have demonstrated that the present invention provides a cannabinoid composition that, on the one hand, promotes the development and maturation of dopamine neurons, prevents their shedding, and improves their viability; and, on the other hand, enhances their ability to secrete dopamine. Therefore, this cannabinoid composition can be used to prepare a drug for treating neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and Lewy body dementia.

[0055] The present invention also provides a pharmaceutical composition for treating neurodegenerative diseases, comprising: the cannabinoid composition of the present invention, and a pharmaceutically acceptable carrier.

[0056] Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A comprehensive description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in compositions may include liquids such as water, phosphate buffered saline, Ringer's solution, physiological saline, balanced salt solution, glycerol, or sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and stabilizers such as albumin. In use, a safe and effective amount of the cannabinoid composition of the present invention is administered to a mammal (e.g., a human). Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician. The precise effective amount for a given subject depends on the subject's size and health status, the nature and severity of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. For a given condition, the effective amount can be determined by routine experimentation, which is within the ability of the clinician to determine.

[0057] The dosage forms of the pharmaceutical composition include oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, atomizers, etc.

[0058] The present invention also provides a drug kit for treating neurodegenerative diseases, comprising: the cannabinoid combination or the pharmaceutical composition of the present invention.

[0059] To facilitate clinical application, the pharmaceutical composition of the present invention can be contained in an injectable device (such as an injection needle), which can contain a single dose of the pharmaceutical composition. The injectable device can be contained in a medicine box for convenient storage and use. The medicine box of the present invention can also include instructions for use to facilitate use by those skilled in the art in the correct manner.

[0060] The experimental process and experimental results of the present invention are described in detail below with reference to the accompanying drawings and examples, thereby illustrating in detail the activation effect of the cannabinoid medicinal composition of the present invention on dopaminergic neuronal cells, which can be used to prepare drugs for the treatment of neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and Lewy body dementia.

[0061] Introduction to the detection proteins related to the examples:

[0062] Nurr1 (nuclear receptor-related protein 1) is a member of the nuclear receptor superfamily of intracellular transcription factors that plays a key role in maintaining the brain's dopaminergic system. Mutations in this gene are associated with disorders related to dopaminergic dysfunction, including Parkinson's disease, schizophrenia, and manic-depressive illness. This protein is thought to be essential for the development of the dopaminergic phenotype in the midbrain.

[0063] Pitx3: Inflammatory factor 3, a protein transcribed from the PITX3 gene. Pitx3 is specifically expressed in brain dopaminergic neurons and plays a key role in the differentiation and maturation of brain dopaminergic neurons. It is considered a transcription factor required for the specific development of midbrain dopaminergic neurons.

[0064] TH: Tyrosine hydroxylase, is a key enzyme in the dopamine biosynthesis pathway. PD is a neurodegenerative disease caused by severe dopamine deficiency in the substantia nigra and striatum. Regulation of TH expression plays an important role in the development and treatment of PD.

[0065] During the differentiation of dopamine neurons, Nurr1 expression first maintains a certain level, followed by TH and Pitx3 expression. Only then can Nurr1 expression induce the expression of marker genes TH and Pitx3 that further promote cell differentiation and maturation. Therefore, to a certain extent, the expression of TH and Pitx3 is more important, and their expression ability can better reflect the vitality of dopamine neurons.

[0066] Example 1

[0067] (1) Experimental process

[0068] 1. Culture medium preparation

[0069] Basal medium 1: DMEM medium, 10% FBS, 1% Penicillin Streptomycin.

[0070] Basal Medium 2: Neurobasal Medium, 1% N-2 Supplement, 1% Penicillin Streptomycin Glutamine.

[0071] Induction Medium 1: Neurobasal medium, 1% N-2 supplement, 1% Penicillin Streptomycin Glutamine, 5% glutamine, 1% B27, 10 ng / ml bFGF, 20 ng / ml EFG, 50 ng / ml GDNF, 2 μM RA; 10 μM SB431542; 200 ng / ml Noggin.

[0072] Induction medium 2: Neurobasal medium, 1% N-2 supplement, 1% Penicillin Streptomycin Glutamine, 1% B27, 10 ng / ml bFGF, 20 ng / ml EFG, 50 ng / ml GDNF, 2 μM RA; 100 ng / ml SHH; 100 ng / ml FGF8b.

[0073] Prepare each culture medium according to the above formula, sterilize the filter membrane after preparation and set aside.

[0074] 2. Cultivation of HDF38 Fibroblasts

[0075] Purchased HDF38 fibroblasts were revived and cultured, then passaged into seven 6-cm culture dishes containing 80,000 HDF38 fibroblasts in Basal Medium 1. Each dish was cultured in a 37°C, 5% CO2 incubator. After three days of culture, the cells were induced to differentiate.

[0076] 3. Induction of Differentiation of HDF38 Fibroblasts

[0077] Experimental Methods: Remove the cells from Basal Medium 1 and wash three times with PBS to remove any residual medium. Then, perform differentiation induction culture: First, replace the cells with Induction Medium 1 containing the test drug, with complete medium changes every 2.5 days for 7 days. After 7 days, replace the culture medium with Induction Medium 2 containing the test drug, with complete medium changes every 3 days for 6 days.

[0078] During the differentiation induction process, cell morphology changes were observed and photographed daily under a microscope (ZEISS AXIO observer). The numbers and culture medium compositions of the seven experimental groups are shown in Table 1 below:

[0079] Table 1 Experimental design of different cannabinoids and their combinations

[0080]

[0081] Note: CBG solution and CBD solution were purchased from SIGMA-ALDRICH, and Δ9-THC was purchased from FUJIFILM.

[0082] 4. Detection of the relative expression levels of DA-related factors

[0083] After 13 days of differentiation induction, cells were harvested and total RNA was extracted from the cells using TRIzol universal reagent (Tiangen Biotech, Beijing, China), and complementary DNA (cDNA) was synthesized. Real-time quantitative PCR was performed using SYBR Green fluorescent dye to detect the relative expression levels of DA-related factors Nurr1, TH-2, and Pitx3 in the cells.

[0084] Among them, the following primers were used to amplify the fragment:

[0085] Primer sequences for Nurr1 gene:

[0086] F: 5'-ACTGCCGATTTCAGAAGTGC-3' (SEQ ID NO: 1),

[0087] R: 5'-CCGGCCTTTTAAACTGTCTGTG-3' (SEQ ID NO: 2);

[0088] Primer sequences for TH gene:

[0089] F: 5'-GAGTACACCGCCGAGGAGATTG-3' (SEQ ID NO: 3),

[0090] R: 5'-GCGGATATACTGGGTGCACTGG-3' (SEQ ID NO: 4);

[0091] Primer sequences for Pitx3 gene:

[0092] F: 5'-AGCACAGCGACTCAGAAAG-3' (SEQ ID NO: 5),

[0093] R: 5'-TTTTTCAGCGAACCGTCCTC-3' (SEQ ID NO: 6).

[0094] qRT-PCR reaction conditions were: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 30°C for 1 minute. After the reaction, the mRNA of the target gene and the internal reference gene were analyzed.

[0095] (2) Experimental results

[0096] 1. Effects of cannabinoid combinations on cell differentiation induction

[0097] The morphology of cells in each culture group was observed under a microscope, and the results are shown in Figure 1: HDF38 fibroblasts have successfully differentiated into dopaminergic neurons, and the cells induced by cannabinoids have differentiated into more dopamine neurons, indicating that cannabinoids can promote the differentiation of HDF38 fibroblasts into dopaminergic neurons; among them, the CBD+CBG group had the largest number of dopaminergic neurons, indicating that the CBD+CBG combination has the strongest promoting effect on the differentiation of fibroblasts into dopaminergic neurons.

[0098] 2. Effects of cannabinoid combinations on the expression of DA-related factors

[0099] As shown in Figure 2, the six groups of cells induced and differentiated successfully expressed dopamine neuron marker genes Nurr1, TH-2, and Pitx3. Among them, the expression levels of Nurr1 in the six induced groups were significantly higher than that in the Cont group, indicating that the HDF38 fiber cells in this experiment have been successfully induced to differentiate into dopaminergic neuron cells (Figure 2A).

[0100] After Nurr1 was successfully expressed, TH-2 and Pitx3 marker genes were subsequently expressed. The expression ability of these two genes can better reflect the survival ability and vitality of dopamine neurons. The results showed that the levels of TH-2 and Pitx3 expressed by the two groups of cells added with CBD+CBG and CBD+THC were significantly better than those of other groups, indicating that compared with single combination drugs, the components of these two drug combinations have a synergistic effect on each other, and have a better promoting effect on the development, maturation and secretion ability of dopaminergic neuronal cells; among them, the level of TH-2 expression in the CBD+CBG group was about 3 times that of CBD+THC, and the level of Pitx3 expression was about 2 times that of CBD+THC. This shows that compared with CBD+THC, CBD+CBG can better increase the activity of dopamine neurons (B and C in Figure 2), and because CBG has no psychoactive effect and is not addictive, the side effects of the drug will be greatly reduced.

[0101] Example 2 Effects of different ratios of CBD+CBG compositions on cell differentiation induction

[0102] Through Example 1, it was found that the combination of CBD+CBG had the best effect in promoting the survival and secretion ability of dopaminergic neurons. However, in Example 1, this was only the effect of a 1:1 mass ratio of CBD and CBG. Therefore, experiments were continued to be designed with different ratios of CBD and CBG to find the best ratio combination for activating dopaminergic neurons.

[0103] (1) Experimental process

[0104] 1. Culture medium preparation

[0105] Basal culture medium 1, basal culture medium 2, induction culture medium 1 and induction culture medium 2 were prepared according to the formula of Example 1, and the filter membranes were sterilized after preparation and set aside.

[0106] 2. Cultivation of HDF38 Fibroblasts

[0107] Purchased HDF38 fibroblasts were revived and cultured, then passaged into 12 6-cm culture dishes containing 80,000 HDF38 fibroblasts in Basal Medium 1. Each dish was cultured in a 37°C, 5% CO2 incubator. After 3 days of culture, the cells were induced to differentiate.

[0108] 3. Induction of Differentiation of HDF38 Fibroblasts

[0109] The above cells were divided into 12 groups for induced differentiation. The experimental method was the same as the relevant operation in Example 1. The design of different ratios of CBD and CBG is shown in Table 2 below, where the amount of CBD added was 1 μg / 10 ml of culture medium.

[0110] Table 2 Experimental design of different ratios of CBD+CBG combinations

[0111]

[0112]

[0113] During the differentiation induction process, cell changes were observed under a microscope (ZEISS AXIO observer) and photographed every day.

[0114] 4. Detection of the relative expression levels of DA-related factors

[0115] After 13 days of differentiation induction, cells were harvested and total RNA was extracted from the cells using TRIzol universal reagent (Tiangen Biotech, Beijing, China), and complementary DNA (cDNA) was synthesized. Real-time quantitative PCR was performed using SYBR Green fluorescent dye to detect the relative expression levels of DA-related factors Nurr1, TH-2, and Pitx3 in the cells.

[0116] (2) Experimental results

[0117] AC in Figure 3 represent the cell morphology of each group on the 4th, 7th and 13th day of induction culture, respectively. The results showed that on the 4th day of induction culture, the production of neurons could be observed in the cells of each culture group, and the number of neurons increased with the increase of CBG in the added drug, indicating that high concentrations of CBG can increase the rate of differentiation of fibroblasts into neurons (A in Figure 3); but after one week of induction culture, the cells of the two groups containing high concentrations of CBG (mass ratio of 1:7 and 1:10) showed different degrees of cell death, and no cell death occurred in the other induced differentiation groups, indicating that high concentrations of CBG can induce drug toxicity and lead to cell death. Therefore, at the CBD concentration in this experiment, the mass ratio of CBD and CBG in the cannabinoid composition is considered safe when it is not higher than 1:5 (B and C in Figure 3).

[0118] Figure 4 shows a comparison of the expression levels of DA-related factors in cells of each group. As shown in Figure 4, when the mass ratio of CBD to CBG was 1:1, 1:3, and 1:5, respectively, the expression levels of TH-2 and Pitx3 were significantly higher than those in the control group and other experimental groups. Among them, the expression levels of TH-2 and Pitx3 were the highest when the ratio was 1:3.

[0119] Example 3 Effects of different ratios of CBD+CBG+CBN compositions on cell differentiation induction

[0120] Through Example 2, it was found that when the mass ratio of CBD and CBG was 1:1 and 1:3, it activated dopaminergic neurons better. Therefore, based on the current two ratios, experiments with different ratios of CBD, CBG and CBN were further designed to find the combination and ratio with the best effect of activating dopaminergic neurons.

[0121] (1) Experimental process

[0122] 1. Culture medium preparation

[0123] Basal culture medium 1, basal culture medium 2, induction culture medium 1 and induction culture medium 2 were prepared according to the formula of Example 1, and the filter membranes were sterilized after preparation and set aside.

[0124] 2. Cultivation of HDF38 Fibroblasts

[0125] Purchased HDF38 fibroblasts were revived and cultured, then passaged into 10 6 cm culture dishes containing 80,000 HDF38 fibroblasts in Basal Medium 1. Each dish was cultured at 37°C in a 5% CO2 incubator. After 3 days of culture, the cells were induced to differentiate.

[0126] 3. Induction of Differentiation of HDF38 Fibroblasts

[0127] The above cells were divided into 10 groups for differentiation induction. The experimental method was the same as the relevant operation in Example 1. The design of different ratios of CBD and CBG is shown in Table 3 below, where the amount of CBD added was 1 μg / 10 ml of culture medium.

[0128] Table 3 Experimental design of different ratios of CBD+CBG+CBN combinations

[0129]

[0130] During the differentiation induction process, cell changes were observed under a microscope (ZEISS AXIO observer) and photographed every day.

[0131] 4. Detection of the relative expression levels of DA-related factors

[0132] After 13 days of differentiation induction, cells were harvested and total RNA was extracted from the cells using TRIzol universal reagent (Tiangen Biotech, Beijing, China), and complementary DNA (cDNA) was synthesized. Real-time quantitative PCR was performed using SYBR Green fluorescent dye to detect the relative expression levels of DA-related factors Nurr1, TH-2, and Pitx3 in the cells.

[0133] (2) Experimental results

[0134] Figure 5 is a comparison of the expression levels of DA-related factors in each group of cells. The results show that adding CBN to CBD and CBG will further enhance the effect of the cannabinoid composition in activating the activity of dopamine nerve cells. Among them, when the mass ratio of CBD and CBG in the CBD+CBG+CBN composition is 1:3, the expression levels of TH-2 and Pitx3 are higher than those in the CBD+CBG+CBN group when the mass ratio of CBD and CBG is 1:1, which once again proves the conclusion of Example 2; further, when the CBD:CBG:CBN (mass ratio) is 1:3:0.3, the expression levels of TH-2 and Pitx3 are the highest, and the effect of activating the activity of dopamine nerve cells is the best.

[0135] Clinical trials were conducted on the composition No. 8 in Example 2 of the present invention, and clinical observations were performed. The specific results are as follows:

[0136] 1. Clinical data

[0137] A total of 14 patients were enrolled, ranging in age from 55 to 93. Five were male and nine were female, with the shortest duration of illness being one year and the longest being approximately 15 years. Ten had Parkinson's disease and four had Alzheimer's disease. Clinical examinations confirmed all cases according to national and industry association standards.

[0138] 2. Treatment Methods

[0139] The drug prepared from the cannabinoid combination No. 8 according to Example 2 of the present invention (sublingual drops, 2 ml per bottle, each bottle containing active ingredients: 10 mg CBD + 30 mg CBG) was administered 1-2 times a day, once 0.5-1 hour before bedtime, or once at noon and before bedtime. This product was taken sublingually, held under the tongue for about 60 seconds, and then swallowed. A course of treatment was 4 weeks, and the dosage for the course of treatment varied depending on the symptoms.

[0140] 3. Efficacy evaluation criteria

[0141] Improvement: The patient, bystanders, or medical tests show that the patient's symptoms have been alleviated, or that their autonomous behavior and consciousness have been improved;

[0142] Significant improvement: Patients, bystanders, or medical tests show a significant reduction in symptoms, or a significant improvement in autonomous behavior and consciousness (e.g., symptoms of Parkinson's disease stage 4 improve to stage 3 symptoms);

[0143] Not obvious: After use, neither the patient himself nor the bystanders nor the medical examinations believe that the patient's symptoms are alleviated or that his autonomous behavior or consciousness is enhanced.

[0144] 5. Treatment outcomes

[0145] Approximately 29% of patients (4 patients) experienced initial improvement in symptoms within the first week of using the drug, 64% (9 patients) experienced improvement within 2 weeks, and 79% (11 patients) experienced significant improvement within the first course of treatment. Currently, approximately 14% of patients (2 patients) have experienced minimal improvement, including one who used the drug for 2 weeks and another who used it for 5 weeks. The improvement rate is approximately 86%.

[0146] No obvious side effects were observed in all patients, and approximately 43% of the patients (6 patients) reported some drowsiness symptoms.

[0147] The case studies presented in this application are preliminary demonstrations of the efficacy of this application. These are only for volunteers. Due to the characteristics of neurodegenerative diseases such as Parkinson's and Alzheimer's, the descriptions of medical history and efficacy are from the patients or their families. These may be subjective or imprecise, but the cases and efficacy are genuine. Since the results of the cases were not described by medical professionals, the technical results of the cases do not represent authoritative medical diagnoses.

[0148] Case 1

[0149] Gao: male, 85 years old, from Rizhao, Shandong, retired doctor, diagnosed with Parkinson's disease in 2017.

[0150] Medical history: Since 2012, the patient has experienced slow movement and expressionless face, which has become increasingly severe. He also has difficulty with fine motor skills, stiff and bent limbs, short steps, constipation, and oily skin on his face and head. Hospitalization and examination in 2017 revealed a mask-like face, a panicky gait, resting tremors in both hands, and hypertonia in his limbs, with a strength level of 5. He was diagnosed with Parkinson's disease. Following four years of medication prescribed by a doctor, the tremor has eased somewhat, but his facial expression has not improved and has gradually worsened. He has also experienced severe drooling, and his walking has become slow and difficult. He cannot walk without a cane and reports being unable to walk.

[0151] Treatment: Starting from July 2021, the patient took the drug prepared from the composition No. 8 of Example 2 of the present invention, once a day before bedtime, one bottle at a time. This has been taken for 5 consecutive weeks.

[0152] Results: After one week of medication, the patient's glazed eyesight improved, his facial expressions became more expressive, he was able to walk indoors, sometimes without a cane, his limb stiffness and curvature improved, and his fine motor skills improved significantly. He regained the ability to cut his nails and peel pills, which had been interrupted for about two years. The patient experienced drowsiness after taking the medication, which he alleviated with tea.

[0153] Case 2

[0154] Xu: Female, 83 years old, from Zhengzhou, Henan Province, diagnosed with Parkinson's disease in 2019.

[0155] Medical History: Since 2014, the patient's movements have gradually slowed, his limbs have become stiff, his sleep time has decreased, and he has developed sleep disturbances. Starting in 2018, his condition worsened significantly, with difficulty walking, speaking, and controlling his tongue. In 2019, he was diagnosed with Parkinson's disease after hospital examinations.

[0156] Treatment: Starting from July 2021, the patient took the drug prepared from the composition No. 8 in Example 2 of the present invention, once a day before bedtime, one bottle at a time. This has been taken for 3 consecutive weeks.

[0157] Results: The patient's self-control began to improve after the second week of treatment. Drooling and uncontrolled tongue movements significantly improved, and the duration of independent standing and walking increased significantly, from less than 10 minutes to over 20 minutes. Sleep time also increased significantly, and mood stabilized.

[0158] Case 3

[0159] Ji Mou: Female, 93 years old, from Heilongjiang, diagnosed with Parkinson's disease in early 2021.

[0160] Medical History: Since 2015, he had experienced stiffness in his limbs and involuntary tremors. In 2020, his condition worsened significantly, leading to his inability to walk independently and prolonged bed rest. He frequently experienced spasms in his limbs, and was unable to perform fine motor tasks such as eating and taking medication. He also developed mood swings and irritability. In early 2021, he was diagnosed with Parkinson's disease by a hospital.

[0161] Treatment: Starting in May 2021, the patient began taking the drug prepared from the composition No. 8 in Example 2 of the present invention, once daily before bedtime, one bottle at a time. This has been taken for 11 consecutive weeks.

[0162] Results: After two weeks of treatment, the patient's limb stiffness was significantly alleviated, the frequency and amplitude of involuntary tremors decreased, and limb spasticity decreased. After 11 weeks of treatment, the patient could stand independently against a wall for nearly five minutes. His speech became clear and his pronunciation correct, and he was able to converse normally. He also experienced increased sleep duration and emotional stability.

[0163] Case 4

[0164] Dai: Female, 81 years old, Malaysian, diagnosed with Alzheimer's disease in 2017.

[0165] Medical History: Since 2014, the patient had experienced severe memory loss, including difficulty opening doors with a key, a tendency to hide things, and paranoia. Starting in 2017, his condition worsened significantly, with cognitive impairment, loss of recognition of family and friends, and inability to remember recent events. Treatment initially focused on non-pharmacological measures, including dietary and lifestyle improvements, supplemented with Donepezil (Aricept). After a year without significant improvement, he switched to herbal remedies and dietary therapy, but the results were still unsatisfactory.

[0166] Treatment: Starting in May 2021, the patient began taking the drug prepared from the composition No. 8 in Example 2 of the present invention, once daily before bedtime, one bottle at a time. This has been taken for 11 consecutive weeks.

[0167] Results: After one week of use, the patient's short-term memory began to improve, and he was able to converse normally with his caregiver. After approximately three to four weeks of use, his memory began to return, and he recognized family members and previously familiar friends. Currently, after 11 weeks of use, the patient's recovery is excellent, and he can recall memories from a year ago. No adverse reactions have been reported.

[0168] Case 5

[0169] Ms. Shen: Female, 68 years old, from Guangdong, diagnosed with Alzheimer's disease in early 2021.

[0170] Medical history: The patient was admitted to the hospital in early 2021 due to "memory loss for more than 3 years and worsening for 1 and a half years." Admission examinations of routine urine and blood tests, seven tumor tests, and quadruple myocardial injury tests showed no obvious abnormalities. Brain CT: multiple lacunar infarctions in the bilateral basal ganglia and corona radiata; white matter degeneration; brain atrophy. Brain magnetic MRI: lacunar infarction foci, white matter degeneration, and brain atrophy. The rest of the examinations showed no obvious abnormalities. The patient was diagnosed with Alzheimer's disease and was treated with drugs such as rivastigmine capsules and memantine for about 4 months without significant improvement.

[0171] Treatment: Starting from May 2021, take the medicine prepared by the composition No. 8 in Example 2 of the present invention, once a day before going to bed, one bottle at a time.

[0172] Results: After three weeks of treatment, the patient's immediate memory improved significantly, and he began to recall events from the past three days and major events from the past week. After 11 weeks, his memory improved significantly, and his difficulty with daily conversation and recalling memories was significantly reduced. He began to sleep during the day and experienced mild drowsiness.

[0173] Case 6

[0174] Mr. Wang: Male, 87 years old, Malaysian, diagnosed with Alzheimer’s disease in 2013.

[0175] Medical History: The patient had been experiencing memory loss and other symptoms since 2007. In 2013, he was diagnosed with Alzheimer's disease and began treatment. Within two years of starting treatment, his memory loss and other cognitive decline had eased, but the effects quickly wore off, and his condition continued to worsen. For over a decade, treatment had somewhat slowed the progression of his symptoms, but it had never led to a significant improvement.

[0176] When we first met him, he was unresponsive to greetings and unable to understand most language, such as medication instructions like "hold under your tongue." He was bedridden or wheelchair-bound, though he could stand, but his mobility was poor and he had little ability to care for himself. He also had poor cognitive function and was unable to recognize his own family members.

[0177] Treatment: Starting from June 2021, take the medicine prepared by the composition No. 8 in Example 2 of the present invention, once a day before going to bed, one bottle at a time.

[0178] Results: After one week of treatment, the patient regained consciousness and understood medication instructions. After nine weeks, he recovered well, chatting freely with his downstairs neighbors and explaining his clothing and shopping. He also began to remember his grandson's phone number, and most normal communication became normal.

[0179] In summary, the present invention uses an experimental method of inducing fibroblast differentiation into a dopamine neuron model to detect the effects of CBD, CBG, THC and their combination on the induction of fibroblast differentiation into dopaminergic neurons. It was found that CBD+CBG has the best promoting effect on the induction and differentiation of dopamine neurons, and can significantly improve the viability and dopamine secretion ability of dopamine neurons. It can be used as a drug for the treatment of neurodegenerative diseases such as Parkinson's disease, and does not contain the addictive substance THC, and the side effects are greatly reduced. Further experiments were designed to combine CBD and CBG in different ratios. It was found that when the mass ratio of CBD and CBG was 1:1 to 1:5, the cannabinoid composition could significantly improve the activity of dopamine neurons, among which the effect was best when the ratio was 1:3. Further experiments were designed to combine CBD, CBG and CBN in different ratios. The results showed that the addition of CBN further enhanced the effect of the cannabinoid composition in activating the activity of dopamine neurons. When the CBD:CBG:CBN (mass ratio) was 1:3:0.3, the effect of activating the activity of dopamine neurons was best.

[0180] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A cannabinoid composition, characterized in that, The cannabinoid composition comprises cannabidiol and cannabidiol, wherein the mass ratio of cannabidiol to cannabidiol is 1:1 to 1:10, 1:0.3 to 1:0.5, 1:0.5 to 1:0.7, or 1:0.7 to 1:

1.

2. The cannabinoid composition according to claim 1, characterized in that, The mass ratio of cannabidiol to cannabidiol is 1:1 to 1:3, 1:3 to 1:5, or 1:5 to 1:

7.

3. The cannabinoid composition according to claim 1, characterized in that, The mass ratio of cannabidiol to cannabinol is 1:

3.

4. The cannabinoid composition according to claim 1, characterized in that, The cannabinoid composition also contains cannabinol.

5. The cannabinoid composition according to claim 4, characterized in that, The mass ratio of cannabidiol to cannabinol is 1:(0.05-1).

6. The cannabinoid composition according to claim 4, characterized in that, The mass ratio of cannabidiol to cannabinol is 1:(0.1-0.5).

7. The cannabinoid composition according to claim 4, characterized in that, The mass ratio of cannabidiol, cannabinol, and cannabinol is 1:3:0.

3.

8. Use of a cannabinoid composition according to any one of claims 1-7 in the preparation of a medicament for treating neurodegenerative diseases.

9. The application as described in claim 8, characterized in that, The neurodegenerative diseases mentioned include at least one of Parkinson's disease, Alzheimer's disease, and Lewy body dementia.

10. The application as described in claim 8, characterized in that, The cannabinoid composition is used to promote the development and maturation of dopamine nerve cells, prevent dopamine nerve cells from detaching, and improve the survival ability of dopamine nerve cells.

11. The application as described in claim 8, characterized in that, The cannabinoid composition is used to enhance the ability of dopamine nerve cells to secrete dopamine.

12. A pharmaceutical composition for treating neurodegenerative diseases, characterized in that, The pharmaceutical composition comprises: the cannabinoid composition according to any one of claims 1-7, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, characterized in that, The dosage forms of the pharmaceutical composition include oils, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, and atomizing agents.

14. A medicine box for treating neurodegenerative diseases, characterized in that, The medicine box includes: The cannabinoid composition according to any one of claims 1-7.