WO2019041778A1

WO2019041778A1 - Application of muscone in preparation of drugs for treating neuroimmune diseases

Info

Publication number: WO2019041778A1
Application number: PCT/CN2018/077989
Authority: WO
Inventors: 王少侠; 胡利民; 闵江; 张玥; 郭虹; 甘国峰
Original assignee: 无锡济民可信山禾药业股份有限公司; 天津中医药大学
Priority date: 2017-09-04
Filing date: 2018-03-05
Publication date: 2019-03-07
Also published as: CN107714679A

Abstract

An application of muscone in preparation of drugs for treating neuroimmune diseases The neuroimmune diseases refer to the neuroimmune diseases caused by microglia-mediated neurotoxic effects, including Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease.

Description

Application of musk ketone in preparing medicine for treating neuroimmune diseases

Technical field

The invention relates to the use of musk ketone, in particular to the use of musk ketone in the preparation of a medicament for treating neuroimmune diseases.

Background technique

Musk ketone is from the genus Moschus berezovskii Flerov or the original Moschus moschiferus L. One of the active ingredients obtained by distillation of the dry secretion of musk in mature male sachet, known as 3-methylpentadecanone, is the main aroma component of musk.

Parkinson's disease is a common dyskinesia disease in middle-aged and elderly people, also known as tremor palsy. The main pathological changes are degeneration and necrosis of dopaminergic neurons in the midbrain and Lewy bodies in the brain. form. The main clinical manifestations are static tremor, muscle rigidity, and retardation. The etiology of Parkinson's disease and the precise mechanism of degeneration of dopaminergic neurons have not yet been fully elucidated. Parkinson's disease is still an incurable progressive disease, and the current treatment is essentially to improve the quality of life and work ability of patients. As the population ages, the incidence of this disease has a clear upward trend. The most conservative estimate is that the incidence of Parkinson's disease in China has increased by at least 20 times in the past 20 years, which is as high as 2%.

At present, Western medicine clinical treatment of Parkinson's disease, mainly using the gold standard drug L-dihydroxy-phenylalanine preparations. However, in the 2-5 years of treatment, many toxic and side effects, such as nausea, anorexia, mild blood pressure, orthostatic hypotension, "end-of-agent phenomenon", "switching phenomenon", mental abnormality, etc., often seriously affect the patient. Obedience and quality of life, with the prolonged taking time, the toxicity and side effects became obvious and serious, and there were problems such as decreased efficacy.

Multiple sclerosis is a central nervous system inflammatory demyelinating disease caused by autoimmune abnormalities. Remission-recurrence is an important feature in the course of the disease and is the most common cause of neurological disability in young adults. The exact etiology and pathogenesis are unknown, but multiple sclerosis is considered to be an autoimmune disease mediated primarily by CD4+ T cells. Currently used drugs for the treatment of multiple sclerosis include immunomodulators and immunosuppressive agents, such as beta interferon (IFN-β), glatiramer, novantrone, etc., but the above The drugs are administered parenterally, the patients are inconvenient to use, and the adverse reactions are large.

Huntington's disease is a hereditary degenerative brain disease, and the cause of Huntington's disease is a mutation in the Huntingtin gene. Although the formation of the corresponding mutant Huntington's protein (Htt) polymer is related to the pathogenesis of Huntington's disease, it is still unclear whether it really causes damage to the nervous system. There is currently no effective method for treating Huntington's disease.

In recent years, with the deepening of research, it has been found that abnormal activation of microglia plays an important role in diseases such as Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis, and Gujarat disease. It is one of the main causes of nerve damage during the pathological development of the above diseases. Microglia are central nervous system immune cells. In the pathological state of Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis, Gujarat disease, etc., small changes in the microenvironment can activate microglia. To quickly move from a resting state to an activated state. The whole activation process presents a waterfall effect: differentiation, proliferation, up-regulation of expression or expression of immune molecules, migration to the site of injury, morphogenesis, immunodominance and functional changes. Its morphologically increased volume, rounded body and deformation, phagocytosis; functionally began to express a large number of membrane surface molecules related to antigen recognition and presentation, such as the expression of major histocompatibility complex II (MHC); Overexpression of matrix protease 9 (MMP9) degrades the blood-brain barrier. More importantly, the secretion levels of many cytokines are significantly up-regulated, and a small part of them such as GDNF promotes neuronal survival, and most of them are tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6). ) and other neuronal damage, as well as chemokines such as monocyte chemoattractant protein 1 (MCP-1), irregular chemokine (Fractalkine), macrophage inflammatory protein 1 (MIP-1) Leukocyte chemotaxis; also produces nitric oxide (NO), reactive oxygen species (ROS) and eicosanoids (such as prostaglandin E2), leading to secondary brain damage. Microglia are not only innate immune effector cells, but also participate in the initiation and regulation of acquired immunity and participate in the pathogenesis of multiple sclerosis.

Therefore, inhibiting the excessive activation of microglia and balancing the various pro-inflammatory factors secreted by it can effectively prevent and treat diseases such as Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis, and Gujarat disease.

Previous literature or patents have also shown that naloxone (J Pharmacol Exp Ther 2000, 293: 607-617), dextromethorphan (J Pharmacol Exp Ther 2003, 305: 212-218), tetrahydroiso Quinoline derivatives (CN101553229A, Chinese Patent Application No. 200780045274.0, published on October 7, 2009), Tripterygium travertine T4 (CN101332200A, Chinese Patent Application No. 200810071485.8, publication date December 31, 2008) Prevents brain inflammation-related diseases by inhibiting microglia activation.

It has been reported in the literature that musk ketone has prevention and treatment of myocardial infarction (China Pharmaceuticals, 2010, 19: 12-13), thrombosis (Journal of Kunming Medical College, 2006, 4: 1-5), and anti-cancer (Phytomedicine, 2010, 17 :63-68) and protect the liver (J Asian Nat Prod Res, 2010, 12: 175-184) and other functions. However, musk ketone has not been reported to inhibit microglia-mediated neurotoxicity and to prevent Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis, and Gujarat disease.

In summary, the abnormal activation of microglia is one of the main causes of neurological damage during the pathological development of Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis, and Gujarat disease. Excessive activation of cytoplasmic cells can effectively prevent and treat the above-mentioned brain inflammatory related diseases. Finding new clinical treatments to treat and/or prevent neuroimmune inflammatory diseases, especially to treat and/or prevent neuroimmune inflammatory diseases associated with microglia, such as treating and/or preventing microglia Neuroimmune inflammatory diseases such as Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis, and Gujarat disease caused by mediated neurotoxic effects are still expected by those skilled in the art.

Summary of the invention

The object of the present invention is to provide a musk ketone in the preparation of a medicament for treating a neuroimmune disease, in particular, a drug for neuroimmune diseases caused by microglia-mediated neurotoxicity, and preparation of musk ketone The resulting drug has an unexpected therapeutic effect on neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat.

To this end, the present invention provides a use of muscone in the preparation of a medicament for treating a neuroimmune disease.

Preferably, the medicament for treating a neuroimmune disease is a medicament for treating a neuroimmune disease caused by a neurotoxic effect.

Preferably, the medicament for treating a neuroimmune disease is a medicament for treating a neuroimmune disease caused by a microglial-mediated neurotoxicity.

Preferably, the medicament for treating a neuroimmune disease is a medicament for treating a neuroimmune disease caused by secretion of an inflammatory medium by microglia.

Preferably, the medicament for treating a neuroimmune disease is a neuroimmune disease drug caused by the treatment of release of microglia NO, TNF-α, and IL-6.

Preferably, the medicament for treating a neuroimmune disease is a neuroimmune disease drug caused by the treatment of lipopolysaccharide-induced release of microglia NO, TNF-α, IL-6.

Preferably, the medicament for treating a neuroimmune disease is a medicament for treating Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease.

Preferably, the medicine for treating neuroimmune diseases is prepared by using musk ketone as a raw material, according to a conventional preparation process, and then adding corresponding auxiliary materials to prepare any oral or injectable dosage form suitable for clinical use.

Preferably, the oral or injectable dosage form comprises a tablet, a granule, a capsule, a pill, a soft capsule, an oral solution, a syrup, a dispersible tablet, an intravenous injection or an intramuscular injection.

Preferably, the excipients include conventional solvents, disintegrants, flavoring agents, preservatives, colorants, binders, lubricants, wetting agents, thickeners, and solubilizers.

The muscone of the present invention is an existing product which is commercially available or can be prepared by the following method:

Compared with the prior art, the present invention provides the use of muscone in the preparation of a medicament for treating a neuroimmune disease, in particular, a neuroglial disease drug caused by microglia-mediated neurotoxicity, The preparation of musk ketone has an unexpected therapeutic effect on neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease.

DRAWINGS

Figure 1 shows the effect of musk ketone on the viability of microglia cells.

Figure 2 shows that musk ketone inhibits the release of NO from microglia by LPS (lipopolysaccharide).

Figure 3 shows that musk ketone inhibits LPS-induced release of TNF-[alpha] from microglia.

Figure 4 shows that musk ketone inhibits LPS-induced IL-6 release from microglia.

Detailed ways

The invention is further illustrated by the following specific examples, which are not to be construed as limiting.

Example 1: Tablet

Taking 1-100g of musk ketone, adding the conventional excipients for preparing tablets, and making 1000 tablets according to the conventional preparation process of tablets, for preventing and treating neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, Encephalitis or Gujarat's disease, 0.1-1 g per tablet, 1-3 tablets each time, 1-3 times a day.

Example 2: Capsule

Take 1-100 g of musk ketone, add the conventional excipients for preparing capsules, mix them, and put them into capsules to make 1000 capsules. For the prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease, each containing muskone 1-100mg, 1-3 capsules per day, 1-day 3 times.

Example 3: Granules

Taking 1-100g of musk ketone, adding conventional excipients for preparing granules, and making 1000 bags according to the conventional preparation process of granules, for preventing and treating neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis Or Gujarat disease, 1-10 grams per bag, 0.5-2 bags each time, 1-3 times a day.

Example 4: Oral solution:

Take 1-100g of musk ketone, add the conventional excipients for preparing oral liquid, and make 1000 pieces according to the conventional preparation process of oral liquid for prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis Or Gujarat disease, each 1ml-10ml, 1-3 each time, 1-3 times a day.

Example 5: Injection

Take 1-100g of musk ketone, add conventional raw materials for injection preparation, and make 1000 injections for prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease , each 5ml-10ml. Adult intravenous or intramuscular injection, 1-3 times a day, once a day.

Example 6: Syrup

Take 1-100g of musk ketone, add the conventional excipients for preparing syrup, and make 1000 pieces according to the conventional preparation process of syrup, for preventing and treating neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis Or Gujarat disease, each 50-250ml, 2-10ml each time, 1-3 times a day.

Example 7

Take 5g of musk ketone, add the auxiliary materials for preparing tablets, and make 1000 tablets according to the conventional preparation process of tablets, for preventing and treating neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease, 2 tablets each time, 2 times a day.

The content of each component of the tablet includes:

5 g of musk ketone, 100 g of starch, 10 g of carboxymethyl cellulose, 10 g of calcium carbonate, and 5 g of talc.

Example 8

5 g of musk ketone was added, and the preparation of the capsule was added, mixed, and filled into capsules to prepare 1000 tablets. For the prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease, 2 capsules each time, 2 times a day.

The content of each component of the capsule includes:

Muskone 5g, pregelatinized starch 80g, microcrystalline cellulose 12g, magnesium stearate 6g, calcium carbonate 5g.

Example 9

Take 5g of musk ketone, add the auxiliary material for preparing granules, and make 1000 bags according to the conventional preparation process of granules, for preventing and treating neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Guca Lei disease, 1 bag each time, 2 times a day.

The content of each component of the granules includes:

5 g of musk ketone, 150 g of lactose, 10 g of polyvinylpyrrolidone, 15 g of tapioca starch, and 5 g of sodium lauryl sulfate.

Example 10:

Take 5g of musk ketone, add the auxiliary material for preparing oral liquid, and make 1000 pieces according to the conventional preparation process of oral liquid for prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Guca Lei disease, 1 each time, 2 times a day.

The content of each component of the oral liquid includes:

5 g of musk ketone, 150 ml of ethanol, 100 g of glucose, 20 g of sodium carboxymethylcellulose, 25 g of polyethylene glycol, and water.

Example 11

Take 5g of musk ketone, add the raw materials for the preparation of injection, and make 1000 injections for the prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat disease, adult vein Or intramuscular injection, 2 each time, once a day.

The content of each component of the injection includes:

Muskone 5g, Tween-80 6g, sodium chloride 50g, sodium carboxymethylcellulose 10g, and water for injection were added to 1000ml.

Example 12

Take 5g of musk ketone, add the auxiliary material for preparing syrup, and make 1000 pieces according to the conventional preparation process of syrup, for prevention and treatment of neuroimmune diseases, especially Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Guca Ray disease, 10ml each time, 2 times a day.

The content of each component of the syrup comprises:

5 g of musk ketone, 200 ml of ethanol, 300 g of sucrose, 20 g of sodium carboxymethylcellulose, 50 ml of peppermint oil, and 10 g of sorbic acid.

Experimental Example 1: Effect of musk ketone on cell viability of microglia cells

The BV-2 mouse microglia cell line was purchased from the Institute of Basic Research of the Chinese Academy of Medical Sciences.

BV-2 cells were cultured in DMEM + 10% FBS + 1% double-antibody culture medium and trypsinized every 3 days. 5% CO2, 95% air, cultured at 37 ° C saturated humidity. The cells in the log phase were harvested and inoculated in a 96-well plate (4×105 cells/mL, 100 μL/well) overnight in a 37°C incubator. The prepared musk ketone (10-5 mol/L) was added and discarded after 24 hours. The supernatant was added to prepare a medium containing 10% CCK-8, incubated at 37 ° C for 30 min, and the absorbance value was read at 450 nm.

The results in Figure 1 indicate that musk ketone does not inhibit cell viability and does not cause cell death.

Experimental Example 2: Effect of musk ketone on the secretion of inflammatory mediators in microglia

The cells in the log phase were harvested and inoculated in a 48-well plate (4×105 cells/mL, 400 μL/well) overnight in a 37°C incubator. The blank control group and the LPS group were incubated with the basic culture medium for 30 min, and the musk ketone was treated. The group was treated with musk ketone (final concentration 10-5mol/L) for 30min, LPS group was added with LPS at a final concentration of 0.1μg/mL, muscone intervention group was added with LPS and musk ketone, and NO and TNF-α were detected after 24h. IL-6 content.

The Griess method detects the reaction of NO:NO ²⁺ with Griess reagent to form a red compound, and the color depth is proportional to the content of NO ³⁺ /NO ²⁺ in the system.

The content of TNF-α and IL-6 was determined by ElISA method: 1 The supernatant of the obtained cell was centrifuged at 10000 g for 15 minutes, diluted 6 times, and used. Add the sample to the sample well, cover the membrane, and incubate for 2 h at room temperature; 2 discard the supernatant, add 300 μl/well to the 1×washing buffer for 5 times, 5 min/time, and finally blot dry with filter paper to add specificity. Primary antibody, cover the membrane, incubate for 2 h at room temperature; 3 discard the supernatant, wash the plate 5 times, 5 min / time; add diluted Biotinylated antibody, cover the membrane, incubate for 1 h at room temperature; 4 discard Clear the solution, wash the plate 3 times; add 100 μl/well to Streptavidin-HRP, cover the membrane and incubate for 20 min at room temperature; 5 discard the supernatant and wash the plate 3 times; add 100 μl/well to TMB, incubate at room temperature for 15 min in the dark; 6 The reaction was terminated by rapid addition of Stop solution (100 μl/well); the detection wavelength was 450 nm and the reference wavelength was 630 nm while reading the plate. 7 A standard curve was prepared based on the measured OD value, and TNF-α, IL-6 content (pg/mL) was calculated.

The results showed that musk ketone could significantly inhibit the release of NO, TNF-α and IL-6 induced by LPS at the concentration of 10 ^-5 mol/L, see Figure 2-4.

Test Example 3: Clinical experiment of musk ketone

METHODS: A total of 350 patients with PD admitted to the Chinese Medicine Hospital from April 2012 to December 2016 were enrolled. The patients were randomly divided into 7 groups: 6 groups in the observation group, specifically in groups 7-12. The muscone-containing preparations prepared in Examples 7-12 were combined with levodopa for treatment of Parkinson's disease in 50 patients in each group; 50 patients in the control group received levodopa for treatment of Parkinson's disease. The clinical efficacy of the 7 groups of patients and the UPDRS score before and after treatment were compared. The results are shown in Table 1.

Table 1 Comparison of UPDRS scores before and after treatment in 7 groups

RESULTS: Studies have shown that musk ketone has an improved effect on motor and affective disorders in patients with (PD). The UPDRS scores of the patients in the groups of Examples 7-12 were significantly lower than those before the treatment and the control group, and the difference was statistically significant (P<0.05).

Claims

The use of a musk ketone in the preparation of a medicament for treating a neuroimmune disease.
The use according to claim 1, characterized in that the medicament for treating a neuroimmune disease is a medicament for treating a neuroimmune disease caused by a neurotoxic effect.
The use according to Claim 1, characterized in that the medicament for treating a neuroimmune disease is a neuroimmune disease drug caused by the treatment of microglial-mediated neurotoxicity.
The use according to claim 1, wherein the drug for treating a neuroimmune disease is a drug for treating a neuroimmune disease caused by secretion of an inflammatory medium by microglia.
The use according to claim 1, wherein the drug for treating a neuroimmune disease is a neuroimmune disease drug caused by the release of microglia NO, TNF-α, and IL-6.
The use according to claim 1, wherein the drug for treating neuroimmune diseases is a neuroimmune disease caused by treatment of lipopolysaccharide-induced release of microglia NO, TNF-α, and IL-6. drug.
The use according to Claim 1, characterized in that the medicament for treating a neuroimmune disease is a medicament for treating Parkinson's disease, multiple sclerosis, Huntington's disease, encephalitis or Gujarat's disease.
The use according to any one of claims 1 to 7, wherein the medicine for treating neuroimmune diseases is prepared by using musk ketone as a raw material, according to a conventional preparation process, and then adding a conventional auxiliary material to prepare any one. Oral or injectable dosage forms suitable for clinical use.
The use according to any one of claims 8 to 4, wherein the oral or injectable dosage form comprises a tablet, a granule, a capsule, a pill, a soft capsule, an oral solution, a syrup, a dispersible tablet, an intravenous injection. Or intramuscular injection.
The use according to any one of claims 8 to 4, wherein the excipient comprises a conventional solvent, a disintegrant, a flavoring agent, a preservative, a coloring agent, a binder, a lubricant, a wetting agent, Thickeners and solubilizers.