Application of pentacyclic triterpenoid compound targeting hydroxysteroid 17-beta dehydrogenase 4

Abstract

The invention provides an application of a pentacyclic triterpenoid compound targeting hydroxyl steroid 17-beta dehydrogenase 4. The pentacyclic triterpenoid compound can effectively relieve or treat central nervous system diseases (inflammatory diseases), including central nervous system degenerative diseases or chronic central inflammatory diseases. The pentacyclic triterpenoid compound can maintain the structural stability or activity of hydroxysteroid 17-beta dehydrogenase and maintain the stability or activity of peroxisome, thereby relieving or treating central nervous inflammation. The pentacyclic triterpenoid compound can target hydroxysteroid 17-beta dehydrogenase 4, inhibit neuroglial cell excessive activation related neuroinflammation, relieve neuron damage or death speed and improve a central nervous system cell metabolism microenvironment.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular, relates to the application of a class of pentacyclic triterpene compounds targeting hydroxysteroid 17-β dehydrogenase 4. Background Art

[0002] Some degenerative diseases of the central nervous system, represented by Alzheimer's disease (AD), are characterized by a comprehensive decline in progressive cognitive function, behavioral ability, and mental state. Patients gradually lose their ability to take care of themselves and even die. They are considered to be one of the most serious global health and social crises in the 21st century. The annual treatment costs of AD patients are high, and the costs of care services and medical insurance cannot be ignored, which imposes a heavy economic burden on patients and society. As the current trend of population aging further intensifies, the incidence of elderly dementia represented by AD has increased year by year, and it has become a major disease and social problem that endangers people's health. Based on the current situation where the growing market demand for AD does not match the production capacity of clinical drugs, and the AD drug development pipeline is single and has an extremely high failure rate, it is imperative to comprehensively explore the occurrence and development mechanism of Alzheimer's disease and find new therapeutic targets and drug development paths.

[0003] As a degenerative disease of the central nervous system, several diseases such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) have Lewy bodies (LB), which are rich in aggregated forms of α-synuclein (α-syn). α-syn is a 14kDa protein without a clear structure, mainly produced in neurons. Under pathological conditions, the monomeric form of the protein gradually forms oligomeric structures and insoluble fibrillar assemblies, which accumulate in the cell in the form of LB. Overexpression or mutation of α-syn leads to progressive defects and loss of dopaminergic neurons in the substantia nigra. Studies have shown that α-syn lesions can be transmitted between cells, leading to disease progression. In microglia, α-syn and mitochondria can be transmitted and degraded through intercellular membrane protrusions, which is a possible reason for cell-to-cell transmission.

[0004] Multiple sclerosis (MS) is an incurable inflammatory autoimmune disease of the central nervous system that affects millions of people worldwide. MS is a chronic demyelinating disease of the central nervous system, the onset and progression of which are driven by a combination of immune dysregulation, genetic susceptibility, and environmental factors. Activation of microglia and astrocytes are key players in the immunopathology of MS, playing specific roles in the anatomical location and stage of the disease and controlling demyelination and neurodegeneration. Laura A. Pasquini et al. believe that the deterioration of myelin debris phagocytosis after microglia depletion reflects the central role of microglia in demyelination. Insufficient phagocytosis subsequently impedes myelin regeneration, especially in myelin-rich areas, and leads to neurodegeneration. Astrocytes are also involved in myelin resorption, especially as an early response to injury that ultimately triggers the recruitment of immune cells. This early response may have a positive or negative impact on lesion pathology, depending on the inflammatory environment, which itself is altered by microglia depletion. Overall, microglia depletion may disrupt the inflammatory status of demyelinating lesions and promote beneficial or harmful responses of astrocytes and oligodendrocytes, thereby affecting neurodegeneration. Therefore, the importance of regulating glial cell-mediated neuroinflammation as a drug target for relapsing-remitting MS is clarified, and drugs that regulate and maintain glial cell homeostasis have great potential in the treatment of multiple sclerosis and other diseases. Summary of the invention

[0005] The purpose of the present invention is to provide the preparation and application of pentacyclic triterpene compounds targeting hydroxysteroid 17-β dehydrogenase 4.

[0006] In the first aspect of the present invention, there is provided the use of a compound represented by formula (I) or its isomer, solvate or precursor, or a pharmaceutically acceptable salt thereof, for preparing a pharmaceutical composition for relieving or treating central nervous system inflammation;

[0007]

[0008] Wherein, R is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen.

[0009] In one or more embodiments, the compound represented by formula (I) or its isomer, solvate or precursor, or their pharmaceutically acceptable salts targets and binds to hydroxysteroid 17-β dehydrogenase 4, thereby maintaining the structural stability or activity of hydroxysteroid 17-β dehydrogenase and the stability or activity of peroxisomes, thereby alleviating or treating central nervous system inflammation.

[0010] In one or more embodiments, the compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, inhibit neuroinflammation associated with excessive activation of glial cells (including microglia), alleviate the rate of neuronal damage or death, and improve the metabolic microenvironment of central nervous system cells by targeting hydroxysteroid 17-β dehydrogenase 4.

[0011] In one or more embodiments, the central nervous system inflammation is central nervous system inflammation with dysfunction of hydroxysteroid 17-β dehydrogenase 4 (including decreased or missing expression, missing or decreased activity, decreased stability, etc.).

[0012] In one or more embodiments, the central nervous system inflammation includes: central nervous system degenerative diseases or chronic central nervous system inflammation.

[0013] In one or more embodiments, the central nervous system degenerative diseases include: Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Huntington's disease (HD), and learning ability or memory impairment.

[0014] In another aspect of the present invention, there is provided the use of a compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, for preparing a pharmaceutical composition for maintaining the structural stability or activity of hydroxysteroid 17-β dehydrogenase 4 (MFE-2) and maintaining the stability or activity of peroxisomes.

[0015] In another aspect of the present invention, there is provided the use of a compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, for preparing a pharmaceutical composition for inhibiting neuroinflammation associated with excessive activation of glial cells, alleviating the rate of neuronal damage or death, or improving the metabolic microenvironment of cells in the central nervous system.

[0016] In one or more embodiments, the pharmaceutical composition further includes donepezil; preferably, the ratio (weight ratio) of the compound represented by formula (I) to donepezil is 10 to 60:1; preferably 15 to 50:1; more preferably 20 to 45:1 (such as 25:1, 30:1, 33:1, 35:1 or 40:1).

[0017] In one or more embodiments, the compound represented by formula (I) has a structure represented by formula (II).

[0018]

[0019] Among them, the English name of the compound of formula (II) is 3-o-α-cyclohexanoyl-11-keto-β-boswellicacid, abbreviated as CKBA (molecular formula is C37H56O5, molecular weight is 581).

[0020] In another aspect of the present invention, a composition (including a cell culture medium) for alleviating or treating central nervous system inflammation is provided, comprising: a compound represented by formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof; preferably, the pharmaceutical composition further comprises donepezil; preferably, the ratio (weight ratio) of the compound represented by formula (I) to donepezil is 10 to 60:1; preferably 15 to 50:1; more preferably 20 to 45:1 (such as 25:1, 30:1, 33:1, 35:1 or 40:1).

[0021] In one or more embodiments, the compound represented by formula (I) or its isomer, solvate or precursor, or their pharmaceutically acceptable salt is used as the main active ingredient (active component) in the composition (including pharmaceutical composition). Preferably, donepezil is also used as the main active ingredient.

[0022] In one or more embodiments, the compound represented by formula (I) or its isomer, solvate or precursor, or their pharmaceutically acceptable salt is used as the only active ingredient (active component) in the composition (including pharmaceutical composition). Alternatively, the compound represented by formula (I) or its isomer, solvate or precursor, or their pharmaceutically acceptable salt, and donepezil are used as the only active ingredients (active components).

[0023] In one or more embodiments, the compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof can also be used in combination with other active ingredients, each exerting an independent effect.

[0024] In one or more embodiments, the compound of formula (I) is prepared by the following method: using 11-carbonyl-β-acetyl boswellic acid as a raw material, replacing its AcO- group with Group.

[0025] In one or more embodiments, the compound of formula (I) is prepared by the following method:

[0026] (i) using 11-carbonyl-β-acetylboswellic acid as a raw material and reacting it with a base to obtain 11-carbonyl-β-boswellic acid; and

[0027] (ii) reacting 11-carbonyl-β-boswellic acid with cyclohexanecarbonyl chloride to obtain a compound represented by formula (I).

[0028] In one or more embodiments, the compound of formula (I) and pharmaceutically acceptable excipients form a pharmaceutical composition. Preferably, the compound of formula (I) is in a systemic / oral dosage form.

[0029] In one or more embodiments, the composition is in an oral dosage form, including suspension or tablet administration.

[0030] In one or more embodiments, the method for preparing an oral dosage form of the compound represented by formula (I) comprises: (1) preparing a suspension of the compound represented by formula (I): using a 0.1-1% sodium carboxymethyl cellulose solution by mass volume ratio for powder suspension preparation, with a final concentration of 10-20 mg / ml, and orally administering at a drug weight ratio of 100±50 mg / kg.

[0031] In one or more embodiments, the preparation method of the oral dosage form of the compound represented by formula (I) includes: (1) preparing tablets of the compound represented by formula (I): selecting diluents: microcrystalline cellulose 101, carboxypropyl methylcellulose and / or starch; selecting three disintegrants: low-substituted carboxypropyl cellulose, cross-linked polyvinylpyrrolidone and / or cross-linked sodium carboxymethyl cellulose, and determining the type and amount of filler and disintegrant based on the drug disintegration time and solubility as indicators; (2) mixing the prepared powder with the diluent and disintegrant evenly, compressing them into large tablets using a single-punch tablet press, and then crushing the large tablets into granules in a mortar, and finally adding the lubricant borosulfate magnesium stearate to the granules through a sieve (such as a 30-mesh sieve) to form the whole granules, and directly pressing the tablets using a single-punch tablet press to obtain tablets.

[0032] In one or more embodiments, the compound represented by formula (I) is dissolved in methanol / DMSO and efficiently passes through the blood-brain barrier. Preferably, a QTRAP 6500plus (SCIEX) mass spectrometer is used to determine the primary and secondary mass spectrometric information of the compound and the corresponding mass spectrometric parameters, thereby determining the quantitative ion pairs, and then using the compound standard test to establish a quantitative method to accurately quantify the compound content and ratio level in brain tissue and plasma.

[0033] In another aspect of the present invention, a medicine box or test kit for alleviating or treating central nervous system inflammation is provided, which comprises the pharmaceutical composition.

[0034] In another aspect of the present invention, a method for alleviating or treating central nervous system inflammation is provided, the method comprising: administering an effective amount of the pharmaceutical composition of the present invention to a subject in need of treatment.

[0035] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 , MFE-2 is a key regulatory target of AD; constructing a conditional knockout of MFE-2 in microglia of AD mice (MFE-2 cKO 5xFAD mice, upper figure) were used for behavioral experiments (middle figure), Aβ and microglia staining, and immunofluorescence staining was used to determine that the early microglial morphology in the brain of AD mice with MFE-2 conditional knockout was significantly abnormal, and the density of cell branched synapses was reduced, indicating that the immune surveillance function and neural support function were limited. As the mice survived to 8 months of age (8m), the number of Aβ plaques in the hippocampus of the brain increased significantly (lower figure), indicating that MFE-2 deficiency caused immune inflammatory dysfunction and aggravated the deposition of Aβ plaques in brain tissue.

[0037] Figure 2 CKBA compounds can effectively penetrate the blood-brain barrier after oral administration. Mass spectrometry was used to evaluate the permeability of CKBA to the blood-brain barrier. Normal mice were given 2.4 mg / mouse (100 mg / kg) of CKBA by oral administration. One hour later, fresh brain tissue and serum were perfused and taken for mass spectrometry detection, and it was found that CKBA can effectively penetrate the blood-brain barrier.

[0038] Figure 3 , CKBA compounds target and bind to MFE-2 protein with high affinity. Studies on the anti-inflammatory effect of CKBA and the pharmacodynamic mechanism of its target have found that CKBA has a very strong affinity with MFE-2.

[0039] Figure 4 , CKBA inhibits microglial inflammatory activation. In vitro cell experiments were conducted to explore the effect of CKBA on microglial inflammatory activation. CKBA was added to LPS-activated BV2 and cultured for 24 hours. It was found that CKBA could significantly inhibit the proliferation of inflammatory microglia, maintain the stability of MFE-2 structure, inhibit protein degradation, maintain peroxisome stability, and play an anti-inflammatory role (Figure A). Figure B is a quantitative analysis of intracellular mitochondrial stress levels.

[0040] Figure 5 CKBA treatment of AD animal models achieved effective relief. AD mice were given CKBA for in vivo intervention studies, and the intervention lasted for 3 months.

[0041] Figure 6The treatment effect of CKBA and donepezil was significantly better than that of CKBA or donepezil alone. CKBA and donepezil were administered orally to AD mice for in vivo intervention. The intervention lasted for 3 months, and the degree of degeneration of the central higher functions such as learning and memory of AD mice was analyzed. The water maze experiment was used to calculate the escape latency and the number of platform crossovers, and the Y maze was used to evaluate the spatial memory and learning ability of mice. DETAILED DESCRIPTION

[0042] The inventors are committed to exploring new molecular therapeutic targets of the pentacyclic triterpenoid compounds described in formula (I). After in-depth research and analysis, the high-affinity functional target of the pentacyclic triterpenoid compounds described in formula (I) was screened, namely, hydroxysteroid 17-β dehydrogenase 4 (MFE-2). At the same time, the inventors' analysis shows that the pentacyclic triterpenoid compounds described in formula (I) can effectively alleviate or treat central nervous system diseases (inflammatory diseases), including: central nervous system degenerative diseases or chronic central inflammatory diseases.

[0043] the term

[0044] The terminology is understood by those skilled in the art. Specifically, the term "alkyl" used herein refers to a straight or branched saturated aliphatic hydrocarbon group containing 1 to 4 carbon atoms (preferably 1 to 2 carbon atoms). For example, alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0045] As used herein, the term "alkenyl" includes straight and branched chain hydrocarbon groups containing at least one carbon-carbon double bond and 2 to 4 carbon atoms, preferably 2 to 3 carbon atoms.

[0046] As used herein, the term "alkynyl" includes straight and branched chain hydrocarbon groups containing at least one carbon-carbon triple bond and 2 to 4 carbon atoms, preferably 2 to 3 carbon atoms.

[0047] The term "halogen" as used herein refers to F, Cl, Br, or I.

[0048] The term "isomer" as used herein includes geometric isomers, enantiomers, diastereomers (eg, cis-trans isomers, conformational isomers).

[0049] The ones used in this article The representation method of is well known to those skilled in the art, which indicates that the group R can be substituted at any one or more substitutable positions on the ring. Moreover, the selection of R can be different at different substitution positions.

[0050] The term "solvate" used herein refers to a compound that carries solvent molecules, for example, the solvate may be a hydrate.

[0051] In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0052] In the present invention, "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0053] In the present invention, "pharmaceutically acceptable carrier" is a pharmaceutically or food-acceptable solvent, suspending agent or excipient for delivering the compound of formula (I), isomer, solvate, precursor, or pharmaceutically acceptable salt thereof to animals or humans. The carrier may be liquid or solid.

[0054] As used in the present invention, the regulation includes "up-regulation" and "down-regulation", wherein "up-regulation" includes but is not limited to: promotion, improvement, elevation, enhancement, etc.; the "down-regulation" includes but is not limited to: reduction, inhibition, antagonism, retardation, blocking, etc.

[0055] Molecular targets

[0056] MFE-2, encoded by the HSD17B4 gene, is a bifunctional enzyme involved in the peroxisomal β-oxidation pathway of fatty acids. It also acts as a catalyst in the formation of 3-ketoacyl-CoA intermediates from straight-chain and 2-methyl branched fatty acids. Defects in this gene affecting peroxisomal fatty acid β-oxidation activity directly lead to the severe central nervous system dysfunction that occurs in D-bifunctional protein deficiency (DBPD).

[0057] Microglia are the most abundant immune cell type in the central nervous system, and the neuroinflammation mediated by them is a common key and important pathological feature and risk factor for a variety of central nervous system degenerative diseases. The inventors found that the expression level of MFE-2 in microglia in the AD mouse model was significantly decreased, and the number of peroxisomes was significantly reduced, which was consistent with the activation of microglia in the brain and the increase of inflammatory factors such as IL-6 and IL-1β. After further specific knockout of MFE-2 in microglia of 5xFAD mice, compared with 5xFAD mice, the morphology of microglia in the brain was abnormal, the immune surveillance function was decreased, and the phenotype of persistent high activation was presented at an earlier age, and the behavioral functions such as memory were significantly reduced. After intervention with pentacyclic triterpenoid compounds (preferably CKBA), the protein level of MFE-2 in microglia can be effectively maintained, the homeostasis of the intracellular environment and the redox balance can be maintained, and the degeneration of learning and memory functions in the individual brain can be slowed down. The research results strongly suggest that MFE-2 functional loss and lipid metabolism disorders can cause the destruction of microglial immune homeostasis, further leading to its dysfunction and aggravating the occurrence and development of neurodegenerative diseases under aging stress.

[0058] Therefore, microglial dysfunction is the key upstream of central immune disorders and neuroinflammation in the process of central nervous system degeneration, and microglial lipid metabolism is an important intervention target for regulating microglial functional phenotypes, alleviating neuroinflammation and disease progression. In particular, the inventors have confirmed that reduced MFE-2 protein content can lead to severe central nervous system disorders, especially when the lack of MFE-2 in microglia leads to persistent central nervous system inflammatory response, leading to aggravated nervous system degeneration and severe cognitive impairment of the nervous system. However, there are currently no drugs targeting MFE-2.

[0059] The present invention discloses for the first time that the pentacyclic triterpene compound (preferably CKBA) binds to MFE-2 with high affinity and can stabilize the intracellular MFE-2 protein level. The compound acts on microglia and effectively controls the central inflammation level by stabilizing the expression of MFE-2, and finally alleviates the progression of neurodegenerative diseases. Therefore, CKBA inhibits central inflammation by targeting microglial MFE-2, and can provide a universal solution for the central nervous immune pathology of neurodegenerative diseases such as AD, PD, and MS, slowing down neurodegeneration, and thus is expected to effectively improve and improve the health level and life treatment of the elderly in society.

[0060] The CKBA can be safely and effectively applied systemically to individuals in need, and can especially efficiently penetrate the blood-brain barrier, and then target the intracellular MFE-2 protein to maintain its normal metabolic function in the cell, stabilize the metabolic microenvironment of glial cells, inhibit over-activation, and significantly reduce the level of inflammatory factors in neurodegenerative diseases, inhibit central nervous system inflammation, and ultimately increase neuronal survival and reduce the accumulation of toxic proteins in the central nervous system. Therefore, it can be used to prepare drugs for the treatment of neurodegenerative diseases, has broad application prospects, and effectively solves the problems of the lack of existing central inflammatory immunomodulatory drugs, insufficient effectiveness, and low safety.

[0061] The pentacyclic triterpene compound can efficiently penetrate the blood-brain barrier and exhibit excellent activity, directly binding to the microglial MFE-2 protein with high affinity, maintaining the balance of mitochondrial metabolism against MFE-2, and significantly inhibiting central nervous inflammation associated with persistent activation of microglia.

[0062] In a preferred embodiment, the "CNS inflammation" refers to a chronic persistent high inflammatory state in the central nervous system mediated by microglia activation, as well as other mechanisms of microglia-astrocyte interaction in neuroinflammation. Microglia, as resident immune cells in the central nervous system, mediate neuroinflammation and play an important role in various physiological and pathological conditions. When neurons are damaged, microglia will quickly initiate an inflammatory response; astrocytes and microglia regulate central nervous system inflammation by secreting a variety of cytokines and inflammatory mediators.

[0063] Pentacyclic triterpenoids

[0064] The present invention includes the compound of formula (I), or its isomers, solvates, precursors, or pharmaceutically acceptable salts thereof, as long as they also have the same or substantially the same function as the compound of formula (I). The "pharmaceutically acceptable salt" refers to a salt formed by the reaction of a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal. These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium) in the form of esters, carbamates, or other conventional "prodrugs". The compound has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereoisomer mixtures, cis or trans isomers.

[0065] The "precursor of a compound" refers to a compound of formula (I), or a salt or solution of a compound of formula (I) which undergoes metabolism or chemical reaction in the patient's body after being taken by an appropriate method.

[0066] As a preferred embodiment of the present invention, the compound has a structure shown in formula (II). The English name of the compound of formula (II) is 3-o-α-cyclohexanoyl-11-keto-β-boswellic acid, referred to as CKBA. Its molecular formula C 37 H 56 O5, molecular weight 581.

[0067] Those skilled in the art should understand that, after knowing the structure of the compound of the present invention, the compound of the present invention can be obtained by a variety of methods well known in the art, using known raw materials, such as chemical synthesis or extraction from organisms (such as animals or plants), and these methods are all included in the present invention.

[0068] For example, as a method for preparing the compound represented by formula (I) of the present invention, the method comprises: using 11-carbonyl-β-acetyl boswellic acid as a raw material, replacing the AcO- group thereof with Group. More preferably, the preparation steps include: (i) using 11-carbonyl-β-acetyl boswellic acid (AKBA) as a raw material, reacting with a base (such as KOH) to obtain 11-carbonyl-β-boswellic acid; and (ii) reacting 11-carbonyl-β-boswellic acid with cyclohexanecarbonyl chloride to obtain the compound shown in formula (I). Other methods for preparing the compound of formula (I) are also included in the present invention, for example, using 11-carbonyl-β-boswellic acid (KBA) as a raw material, reacting it with cyclohexanecarbonyl chloride to directly obtain the compound shown in formula (I).

[0069] The synthesized compound can be further purified by column chromatography, high performance liquid chromatography, etc. In addition, it can also be further purified by crystallization. It should be understood that a variety of purification methods can be applied to the present invention.

[0070] In a preferred embodiment of the present invention, a method for preparing a pharmaceutical composition using the pentacyclic triterpene compound is provided, comprising the following steps: (1) preparing the pentacyclic triterpene compound; (2) mixing the pentacyclic triterpene compound suspension with a sodium carboxymethyl cellulose solution having a mass volume ratio of 0.1-1% to prepare the pentacyclic triterpene compound powder suspension, with a final concentration of 10-20 mg / ml. Preferably, for example, the pentacyclic triterpene compound is orally administered at a drug weight ratio of 100±50 mg / kg.

[0071] In a preferred embodiment of the present invention, a tablet of the pentacyclic triterpene compound is prepared: wherein (1) the pentacyclic triterpene compound suspension is prepared by mixing the pentacyclic triterpene compound powder with a sodium carboxymethyl cellulose solution having a mass volume ratio of 0.1-1%, and the final concentration is 10-20 mg / ml, and the drug is orally administered at a drug weight ratio of 100±50 mg / kg; (2) the tablet of the pentacyclic triterpene compound: three diluents are selected: microcrystalline cellulose 101, carboxypropyl methyl cellulose, and starch; and three disintegrants are selected: low-substituted carboxypropyl cellulose, cross-linked polyvinylpyrrolidone, and cross-linked sodium carboxymethyl cellulose. The type and amount of filler and disintegrant are determined based on the disintegration time and solubility of the drug; (3) the prepared pentacyclic triterpene compound powder is mixed evenly with the diluent and disintegrant, compressed into large tablets using a single-punch tablet press, and then the large tablets are crushed into granules in a mortar, and finally the lubricant borokonium stearate is added to the granules, sieved through a 30-mesh sieve, and directly tableted using a single-punch tablet press to obtain the pentacyclic triterpene compound tablets.

[0072] In a preferred embodiment of the present invention, a pharmaceutical composition for treating central nervous system degenerative diseases is provided, wherein the pharmaceutical composition comprises an effective amount of the pentacyclic triterpene compound and a pharmaceutically acceptable carrier.

[0073] In a preferred embodiment of the present invention, the pentacyclic triterpene compound is administered systemically, preferably orally, providing the operational convenience of long-term drug control of environmental conditions and the therapeutic flexibility of quantitative and adjustment of treatment courses, while providing the possibility of dynamic detection of drug concentrations and adverse reactions.

[0074] In a preferred embodiment of the present invention, the in vivo dosage of the compound is 50-150 mg / kg.

[0075] In a preferred embodiment of the present invention, the compound is introduced into the body of a subject in need thereof by oral administration / oral administration / feeding / mixing with drinking water / mixing with food.

[0076] In a preferred embodiment of the present invention, the subjects treated with the compound include humans and non-human mammals.

[0077] In a preferred embodiment of the present invention, the in vitro experimental intervention concentration of the compound is 1-10 μM.

[0078] In a preferred embodiment of the present invention, the compound is dissolved in DMSO to prepare a stock drug, which is then diluted according to different dilution gradients and added to the culture medium of the desired treated cells.

[0079] In a preferred embodiment of the present invention, a 0.1-1% volume-to-mass ratio sodium carboxymethyl cellulose solution is used to prepare a suspension, the drug concentration is 15 mg / mL, and the drug is administered by gavage, and the individual dosage is 50-150 mg / kg / day.

[0080] In a preferred embodiment of the present invention, the pentacyclic triterpene compound is formulated in a diet / feed and administered with meals. It should be understood that the physician may also make appropriate dosage changes according to the actual clinical situation.

[0081] In a preferred embodiment of the present invention, the pentacyclic triterpene compound is dissolved in DMSO / methanol or the like to prepare a suspension, and / or a medicated feed and / or an oral tablet.

[0082] In a preferred embodiment of the present invention, a preferred highly lipophilic small molecule compound is provided, which can more preferably effectively penetrate the brain barrier and be enriched in the target organ brain tissue to exert a therapeutic effect.

[0083] In a preferred embodiment of the present invention, the therapeutic target cells are mammalian cells, and the mammals include humans and non-human mammals.

[0084] In a preferred embodiment of the present invention, the therapeutic target cells are selected from the following groups: glial cells (including microglia, astrocytes, oligodendrocytes, ependymal cells, radial glial cells, satellite cells, Schwann cells, intestinal glial cells, etc.) and various types of neuronal cells, or a combination thereof.

[0085] In a preferred embodiment of the present invention, the compound or combination is used to treat aging / injury related degenerative diseases.

[0086] In a preferred embodiment of the invention, the compound or combination is used to treat a neurodegenerative disease.

[0087] In a preferred embodiment of the present invention, the compound or combination is used to inhibit the level of central inflammation and / or promote the clearance of neurotoxic proteins and / or the survival of neural clouds, thereby alleviating the degree of decline in brain learning and memory functions;

[0088] In a preferred embodiment of the present invention, the central nervous system degenerative diseases include any age-related degenerative diseases of the central nervous system such as AD, MS, PD, etc., accompanied by long-term and persistent activation of central inflammatory response.

[0089] The pentacyclic triterpene compound oral dosage form disclosed in the present invention is simple to synthesize, has good stability, low cost, no immune rejection, can specifically inhibit the differentiation of microglia in an inflammatory environment, has a very significant therapeutic effect, is not prone to disease recurrence after treatment, and has few side effects.

[0090] In some embodiments, the pentacyclic triterpenoid compound can be connected or combined with a functional molecule. For example, the functional molecule is a marker with a tracing function, including but not limited to fluorescent dyes, MRI contrast agents, radioactive imaging agents, magnetic particles or chemical reagents with a coloring function. For example, the marker with a tracing function or a functional small molecule can be fluorescein isothiocyanate (FITC).

[0091] In some embodiments, the functional molecules are functional small molecules, including inorganic small molecules and organic small molecules, and their molecular weight is less than 1000 Daltons.

[0092] In some embodiments, the functional molecules are preparations with molecular packaging and cargo carrying functions, including but not limited to: liposomes, polymers, dendritic molecules, nano-packaging preparations, etc.

[0093] The connection mode of the small analytical compound CKBA described in the present invention and the functional molecule can be covalently connected or non-covalently connected. It should be understood that as long as the activity of the small molecule compound and the function of the functional molecule can be retained, any connection mode can be included in the present invention. Covalent connection usually connects two molecules in a way of forming a covalent bond, and some non-covalent connections (not forming a covalent bond), such as coupling, adsorption, combination, etc., can also be applied. As a preferred embodiment of the present invention, the polypeptide and the functional molecule are connected by a chemical bond; more preferably, the chemical bond is a peptide bond.

[0094] Combination medication

[0095] The present invention provides a method for combined medication, comprising a method for using the pentacyclic triterpene compound in combination with donepezil.

[0096] The present invention provides the use of the pentacyclic triterpene compound and donepezil (in combination) for preparing a mixture, a pharmaceutical composition or a medicine kit for relieving or treating central nervous system inflammation.

[0097] When administering, the pentacyclic triterpene compound can be administered first, followed by donepezil; or the order can be reversed; or the administration can be performed simultaneously. It should be understood that a variety of administration methods are included in the present invention.

[0098] The invention provides a mixture of small molecule compounds, which contains: the pentacyclic triterpene compound and donepezil as active components.

[0099] The present invention provides a pharmaceutical composition, comprising: (a) an effective amount of the pentacyclic triterpene compound; (b) an effective amount of donepezil; and (c) a pharmaceutically acceptable carrier or excipient.

[0100] The pharmaceutical composition or mixture of the present invention can be prepared into any conventional preparation form by conventional methods. The dosage form can be various, as long as it can effectively allow the active ingredient to reach the body of a mammal. For example, it can be selected from: injection, infusion, tablet, capsule, pill. The active ingredient can be present in a suitable solid or liquid carrier or diluent.

[0101] The mixture or pharmaceutical composition of the present invention can also be stored in a sterile apparatus suitable for injection or instillation. Generally, the pentacyclic triterpene compound and donepezil as active ingredients in the pharmaceutical composition of the present invention can account for 0.01-20% of the total weight of the pharmaceutical composition, and the rest can be a pharmaceutically acceptable carrier.

[0102] The effective dosage of the pentacyclic triterpene compound and donepezil used may vary depending on the mode of administration and the severity of the disease to be treated. When necessary, the pentacyclic triterpene compound and donepezil may also be administered in combination with other active ingredients or drugs.

[0103] The present invention also provides a medicine box for alleviating or treating central nervous system inflammation, wherein the medicine box comprises: a container 1, and the pentacyclic triterpene compound placed in the container 1; and a container 2, and donepezil placed in the container 2.

[0104] The pentacyclic triterpene compound and donepezil are both small molecule compounds, so the medicine kit may also contain a mixture of the pentacyclic triterpene compound and donepezil, wherein the contents of the pentacyclic triterpene compound and donepezil are as described above.

[0105] In addition, the medicine box may also contain some auxiliary medication materials, such as injection syringes, etc.

[0106] In addition, the medicine box may also contain instructions for use, explaining the method of using the combined medication method of the present invention to relieve or treat central nervous system inflammation.

[0107] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0108] Materials and methods

[0109] 1. Preparation of CKBA in vivo and in vitro drugs and dosage forms

[0110] The use of CKBA compounds or their derivatives is systemic medication. CKBA (molecular formula: C37H56O5, molecular weight: 580.85, solubility: fat soluble, soluble in methanol / DMSO, insoluble in water, self-synthesized). CKBA is a small molecule compound obtained by structural modification and optimization of the active natural product molecule AKBA as the lead compound. The specific synthesis involves AKBA as the starting material, hydrolysis reaction under alkaline conditions to obtain KBA intermediates, KBA cyclohexanecarboxylation reaction and purification to obtain CKBA compounds. The detailed synthesis route is as follows:

[0111]

[0112] In vivo administration: 0.1-1% volume-to-mass ratio of sodium carboxymethyl cellulose solution is used to prepare a suspension, the drug concentration is 15 mg / mL, and the drug is administered by gavage, and the individual dosage is 50-150 mg / kg / day. Preferably, CKBA is configured in diet / feed and administered with meals, and the individual dosage is 50-150 mg / kg / day.

[0113] Cell treatment: CKBA was dissolved in DMSO for cell treatment. The storage solution concentration was 5mM (1000X) and stored at -20°C. When used, the storage solution was added to the cell culture medium at a ratio of 1:1000 and mixed evenly. The final concentration of the working solution was 5μM. The cells were treated for 24 to 48 hours and then collected for subsequent experiments.

[0114] 2. Build MFE-2 cKO 5xFAD, 5xFAD, MFE-2 cKO 、MFE-2 cKI 5xFAD mice, exploring the regulatory effect of MFE-2 on microglia function

[0115] CRISPR-mediated homologous recombination was used to insert the same LoxP site into the mouse Hsd17b4 gene to construct Hsd17b4fl / fl mice, which were then bred with Cx3cr1Cre mice to obtain Cx3cr1CreHsd17b4fl / fl (MFE-2cKO).

[0116] Cx3cr1CreHsd17b4 was obtained by breeding Hsd17b4fl / fl with Cx3cr1Cre and 5xFAD mice (Alzheimer's disease mice). fl / fl 5xFAD(MFE-2 cKO 5xFAD).

[0117] Take Hsd17b4 from the same litter fl / fl control) and Hsd17b4 fl / fl5xFAD mice were used as a control for normal expression of MFE-2. At the same time, the exogenous mHsd17b4 gene fragment was inserted into the mouse H11 locus using the CRISPR method. fl / fl MFE-2 was obtained by breeding with Cx3cr1Cre and 5xFAD mice. cKI 、MFE-2 cKI 5xFAD).

[0118] The regulatory effect of MFE-2 on Microglia homeostasis and inflammatory activation process was observed in mice at different stages.

[0119] 3. Immunofluorescence imaging analysis of microglia-mediated central nervous system inflammation under different animal model conditions

[0120] According to the progression of central nervous system inflammation and the distribution characteristics of microglia, MFE-2 was injected into the glioma cells at the early stage (2 months), the middle stage (4 months), and the late stage (8 months). cKO 5xFAD, 5xFAD, MFE-2 cKO The mice and control mice were euthanized, and the blood in the brain tissue was completely removed by cardiac perfusion. After replacing the tissue fixative with systemic perfusion fixation, the intact brain tissue was collected and placed in the fixative for 24 hours, washed with PBS and dehydrated with gradient sucrose solution, embedded in OCT and made into 30 μm thick tissue sections, and stained with immunofluorescence to analyze the expression levels of MFE-2, Iba-1, Cx3cr1, TNFα, F4 / 80, and IL-6.

[0121] Among them, the immunofluorescence staining steps mainly include incubation with 10% goat serum blocking solution, incubation with primary and secondary antibodies, and DAPI nuclear staining. After sealing, a Leica SP8 laser scanning confocal microscope was used to photograph the distribution and morphological characteristics of microglia; ImageJ software was used for microglia morphological analysis and quantitative analysis of intracellular fluorescence signal intensity.

[0122] 4. Animal experiment and mass spectrometry method of CKBA penetrating the blood-brain barrier

[0123] CKBA absolute quantitative detection experiment mass spectrometry standard solution and standard curve: CKBA standard methanol dilution (10ng / mL, 1ng / mL), preparation of standard curve: use methanol to prepare CKBA standard solutions with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500ng / mL. After centrifugation of the above solutions (18800g, 15min, 4℃), take 50μL of the supernatant and put it into a sample bottle for mass spectrometry analysis; preparation of spiked blank serum standard curve (Spiked standard): take 10μL of CKBA working solution of each concentration in the above CKBA standard methanol dilution into 90μL of blank serum and vortex for 30s.

[0124] Preparation method of test samples: Treatment of blank serum samples (Double Blank): Take 10 μL of methanol in 90 μL of blank serum and vortex for 30 seconds; Treatment of actual serum samples: The above-mentioned blank serum standard curve solution, blank serum samples and actual serum samples were precipitated with 3 times methanol and vortexed for 30 seconds; after centrifugation (18800g, 15min, 4℃), 50 μL of supernatant was taken and placed in a sample bottle for mass spectrometry analysis; 1 mL of methanol was added to each tube of all brain tissue homogenates (6+2), vortexed thoroughly and centrifuged (18800g, 15min, 4℃), and the supernatant was taken and centrifuged again for use; Spiked blank brain tissue homogenate standard curve (Spiked Preparation of the CKBA standard: take 10 μL of each concentration of CKBA working solution in the above-mentioned CKBA standard methanol dilution solution into 90 μL of blank brain tissue homogenate supernatant, and vortex for 30 seconds; treatment of the blank brain tissue homogenate sample: take 50 μL of the blank brain tissue homogenate supernatant and put it in a sample bottle for mass spectrometry analysis; treatment of the brain tissue homogenate actual sample: take 50 μL of the actual brain tissue homogenate supernatant and put it in a sample bottle for mass spectrometry analysis.

[0125] Detection method and process: The primary and secondary mass spectrometry information and corresponding mass spectrometry parameters of CKBA were determined using QTRAP 6500plus (SCIEX) mass spectrometer to determine the quantitative ion pair. The standard used a methanol diluted solution of CKBA standard (5 μg / mL). The instrument used a mass spectrometer QTRAP 6500plus (SCIEX). The mass spectrometry conditions were direct injection by syringe pump (10 μL / min), Q1 MS scanning, positive ion mode, scanning range: m / z 200-800, Gas1:20; Gas2:0; Curtain Gas:20; Temperature:0; ISVF:5500; DP:80, and / or Product Ion scanning, positive ion mode, specifying the parent ion. The parent ion obtained from the primary mass spectrometry result was 581.6. The detection parameters included Gas1:20; Gas2:0; Curtain Gas:20; Temperature:0; ISVF:5500; DP:80. Gas:20; Temperature:0; ISVF:5500; DP:80; CE:50.

[0126] 5. Pull-down and Western blot experiments of affinity of CKBA-MFE-2 direct binding

[0127] CKBA Pull-dwon detects the direct interaction between CKBA and MFE-2. Specifically, the candidate small molecule drug CKBA was labeled with Biotin, CKBA-biotin was added to the culture medium (final concentration 5 μM) for 6 hours, the cells were washed and lysed, and incubated with Streptavidin magnetic beads at 4°C for 3 hours under rotation. After washing the beads 4 times, the loading buffer was added and boiled at 96°C for 5 minutes. The MFE-2 protein level was detected by Western blot (WB) to evaluate the direct interaction between the small molecule compound and MFE-2.

[0128] The Western blot protein immunoblotting detection steps include: immersing fresh cells and tissue samples in lysis buffer, placing them on ice for 10-15 minutes, and after observing that the cells and tissues are fully lysed into a homogeneous turbid liquid, placing them in an ice bath for 10 minutes, centrifuging at 12,000 rpm for 10 minutes, taking the supernatant, and using the BCA method to detect the total amount of protein, and then performing the steps of loading, electrophoresis, membrane transfer, blocking, primary antibody, secondary antibody incubation, etc. After development with ECL luminescent developer, use ImageJ to analyze the protein content value.

[0129] 6. CKBA treatment of LPS activated BV2 for CCK-8 experiment

[0130] Mouse microglial cell line BV2 was inoculated into 96-well flat-bottom plates and cultured overnight to allow the cells to fully adhere to the wall. LPS was used to stimulate and activate BV2. LPS (Sigma) storage solution (5 mg / mL) was added to the supernatant of BV2 cell culture medium and mixed. Cell morphology was observed after activation for 12 hours. CKBA working solution was further prepared according to the above method, and CKBA was added to the cell culture medium at a final concentration of 5 μM and cultured for 6 to 24 hours.

[0131] Then, CCK8 (Japanese colleagues) was used to detect cell viability. The cell culture medium of the 96-well plate was removed, and 100ul of the diluted CCK8 reaction solution was added to the well plate. Pay attention to protecting 100uL from light; incubation conditions: 37°C incubator, about 1-2 hours; after incubation for 1 hour, the cells were taken out and the cell color (orange) signal was detected using a multifunctional microplate reader (Tecan Infinite 200pro, Austria); according to the signal intensity, the cells can be taken out after 1.5 / 2 hours of incubation, and the changes in the cell color (orange) signal can be detected at multiple points; the color intensity value was recorded, and SPSS was used for statistical analysis and drawing.

[0132] 7. Seahorse experiment of CKBA treating LPS-stimulated BV2

[0133] The specific methods of CKBA treatment and LPS activation of BV2 cell line are as described above.

[0134] The main steps of the Seahorse experiment include: Seahorse XF cell mitochondrial stress analysis: Use the above experimental method to obtain single cells of brain tissue, collect microglia in brain tissue by flow sorting, inoculate primary microglia into Seahorse XF cell culture plates 4 to 7 days in advance, and after complete attachment, use double distilled water to hydrate the probe plate one day in advance and place it in a 37°C incubator without CO2 overnight. The next day, replace it with calibration solution to further balance the hydration probe. On the day of the experiment, prepare the detection solution, add the corresponding substrates of the kit such as glucose, pyruvate, glutamine, etc., and adjust the pH. 7.4. Wash the cells three times with the detection solution, replace the cell culture solution with the detection solution, and place them in a CO2-free 37°C incubator for testing within 1 hour. Prepare the detection drugs Oligo, FCCP, ROT / AA and add them to the probe plate drug addition holes A, B, and C. Then, run the probe plate hydration plate and perform the calibration process. Then remove the hydration plate and place the probe plate on it to start the test. Record the OCR / ECAR after drug addition at different time points. After recording, remove the cell plate and use an inverted microscope to take pictures and count the number of microglia in each well. After averaging the OCR / ECAR according to the number of cells, quantify the mitochondrial stress level in the cells.

[0135] 8. CKBA oral preparation drug treatment plan

[0136] Administration is by gavage and / or with feed and / or by oral tablet.

[0137] In the animal model treatment effect evaluation study, 15 mg / mL CKBA suspension (solvent 0.5% sodium carboxymethyl cellulose) was administered by gavage with a dosing volume of 200 ul (equivalent to a dose of 100 mg / kg) for 5 consecutive months, once every other day. The treatment effect was evaluated after the end of the dosing cycle.

[0138] 9. Mouse models of neuroinflammation and degenerative diseases

[0139] (1) 5xFAD transgenic mice, which overexpress mutant human APP (695) and human PS1 with Swedish (K670N, M671L), Florida (1716V), and London (V717I) familial Alzheimer's disease (FAD) mutations and two FAD mutations (M146L and L286V). The expression of both transgenes is regulated by the mouse neurospecific regulatory element Thy1 promoter to drive overexpression of the transgene in the brain. Mice of this strain have high APP expression, which is associated with a high load and accelerated accumulation of the 42 amino acid long beta-amyloid protein (Aβ-42) species. 5XFAD mice produce almost exclusively Aβ-42, which accumulates rapidly in the brain.

[0140] (2) Preparation of MS mouse (EAE) model and evaluation of CKBA intervention effect

[0141] 10-week-old C57BL / 6 female mice were immunized with MOG35-55 emulsion, and PTX was given on the day of immunization and the next day, respectively. Different doses of the small molecule drug FenCKBA were orally administered 12 days after immunization. The percentage of body weight change of each group of animals was recorded, and the clinical manifestations of each group of animals were scored. MOG35-55: myelin oligodendrocyte glycoprotein polypeptide 35 and 55. PTX: pertussis toxin. Fingolimod: fingolimod.

[0142] (3) Preparation of PD mouse model and evaluation of CBKA intervention effect

[0143] In SPF Balb / C mice, healthy, male, 4-6 weeks, weighing 18g-20g, MPTP 20mg / kg / d was injected intraperitoneally for 14 consecutive days. The control group used an equal volume of normal saline for intraperitoneal injection, and the operation and precautions were the same. After the MPTP administration, the model was successfully established. The corresponding CKBA drug or control solvent was given; the normal group and PD model group used an equal volume of PBS for intraperitoneal injection, and the operation and precautions were the same.

[0144] (4) Evaluation of therapeutic effects in mouse models, behavioral experiments (rotarod, water maze, open field)

[0145] Evaluation of the therapeutic effect of CKBA on AD: 5xFAD mice aged 8-10 months, 5xFAD mice treated with the small molecule drug CKBA, and control mice were selected, and behavioral experiments including water maze, open field, and rotarod were performed to evaluate the higher functions of the central nervous system. The behavioral experiment process was fully recorded and analyzed offline using an animal motion trajectory tracking system (EthoVison XT 16.0).

[0146] Morris water maze: The mice were subjected to 5 days of adaptive training, abnormal individuals were excluded, and a probe test was conducted on the last day after the platform was removed. Quantitative data such as the platform latency after each quadrant of the mice entered the water during the learning period, the number of times the platform area was crossed in the last test, and the latency to enter the platform area were recorded;

[0147] Open field test: record the spontaneous activity path of mice in each area in a strange open field of 50cm*50cm*40cm for 5 minutes, analyze the movement distance and time of mice in the central area to evaluate the anxiety changes of mice;

[0148] Rotarod test: The mice were subjected to three adaptive trainings and then to continuous tests, each lasting 5 minutes. The latency of the mice to fall off the rotarod was recorded to evaluate the motor function of the mice's nervous system.

[0149] Example 1: Effect of MFE-2 knockout on pathological plaque deposition in the brain of AD animals

[0150] The inventors conditionally knocked out MFE-2 in microglia of AD mice (MFE-2 cKO 5xFAD mice; referred to as ADcKO) Figure 1 above) for analysis.

[0151] Comparison of 5xFAD and MFE-2 by behavioral experiments cKO Differences in higher-order central nervous system activity between the two groups of 5xFAD mice. Figure 1 The middle figure shows that the learning and memory abilities of the knockout group mice were significantly reduced compared with the control AD ​​mice, and the average anxiety levels were significantly increased.

[0152] Further staining of Aβ and microglia revealed that the early microglial morphology in the brain of AD mice with conditional knockout of MFE-2 was significantly abnormal, and the density of cell branched synapses decreased, indicating that the immune surveillance function and neural support function were limited. As the mice survived to 8 months of age, the number of Aβ plaques in the hippocampus of the brain increased significantly, indicating that MFE-2 deficiency caused immune inflammatory dysfunction and aggravated the deposition of Aβ plaques in brain tissue ( Figure 1 ).

[0153] Therefore, MFE-2 deficiency causes a significant decrease in microglial function, leading to the progression of AD and aggravating functional impairment of the central nervous system such as learning and memory.

[0154] The above results indicate that MFE-2 knockout causes a significant increase in pathological plaque deposition in the brains of AD animals, and MFE-2 is a key therapeutic target for effectively inhibiting Aβ deposition.

[0155] Example 2: Penetration of CKBA through the blood-brain barrier

[0156] Whether CKBA can effectively alleviate AD is an exploration with great potential clinical value. The inventors used mass spectrometry to evaluate the penetration of CKBA into the blood-brain barrier. First, an effective method for absolute quantification of CKBA by mass spectrometry was established. According to the aforementioned optimized chromatographic mass spectrometry method, CKBA standard was detected. First, 10 ng / mL of CKBA was sampled and it was found that a higher characteristic chromatographic peak eluted at about 4.52 min. The concentration of the CKBA standard was reduced and the sample was continued to be analyzed (1 ng / mL). It was found that the characteristic chromatographic peak could still be observed at about 4.52 min, and its response was about 1 / 10 of that of 10 ng / mL of CKBA. The standard curve results showed good linearity and detection accuracy, indicating that the concentration of CKBA in methanol solution can be more accurately quantified using the established quantitative method. The parent ion obtained by the primary mass spectrometry scan was 581.6, that is, CKBA added a proton in the mass spectrum; the secondary mass spectrometry scan obtained the corresponding daughter ion fragment spectrum, and the same quantitative daughter ion 407.5 selected by the company's previous test was selected. Thus, the quantitative ion pair of CKBA was 581.6 / 407.5 ( Figure 2 ).according to Figure 2 B, The established CKBA quantitative method is specific and the standard curve has good linearity.

[0157] The absolute content of CKBA in brain tissue was further detected based on the above-mentioned optimized method. The absolute content of CKBA in brain tissue was detected by gavage of normal mice with CKBA for 1 hour, followed by perfusion and collection of fresh brain tissue and serum for mass spectrometry detection.

[0158] The results showed that CKBA can effectively penetrate the blood-brain barrier ( Figure 2 ).

[0159] Therefore, CKBA can effectively cross the blood-brain barrier, which is an important basis for the systemic use of CKBA to treat neurodegenerative diseases.

[0160] Example 3: CKBA directly binds to MFE-2 in living cells to protect the stability of MFE-2 in cells CKBA (molecular formula: C37H56O5, molecular weight: 580.85, solubility: fat soluble, soluble in methanol / DMSO) ( Figure 3 A). CKBA is a small molecule compound obtained by structural modification and optimization of the active natural product molecule AKBA as the lead compound.

[0161] The inventors found that CKBA has a very strong affinity with MFE-2, KD = 0.97nM ( Figure 3 B).

[0162] In vitro experiments and pull-down assays showed that CKBA could bind to MFE-2 protein in microglial cell line (BV2). Figure 3 C).

[0163] The effect of CKBA on MFE-2 protein was further explored. The inventors used CKBA to treat normal BV2 cells and found that CKBA did not affect the expression of MFE-2 in the cell baseline state by detecting protein expression. The expression of MFE-2 in BV2 cells was significantly downregulated after LPS stimulation. Treatment with CKBA can effectively maintain the expression and content of MFE-2 in cells ( Figure 3 DE).

[0164] Based on the above results, MFE-2 has an effective protective effect on the prognosis of AD neurodegenerative diseases, and MFE-2 can effectively maintain intracellular MFE-2 expression.

[0165] Example 4: Systemic administration of CKBA targeting MFE-2 effectively improves the pathological process of neurodegenerative changes in AD mice and exerts a therapeutic effect

[0166] 1. CKBA regulates microglial activation by targeting MFE-2

[0167] Based on the above results, it was found that MRE-2 is closely related to the activation of AD microglia. In order to further clarify the regulatory effect of CKBA targeting MFE-2 on microglial activation, as well as its effect on central inflammation and brain function prognosis in AD, in vivo and in vitro CKBA intervention studies were conducted.

[0168] In vitro cell experiments analyzed the effect of CKBA on microglial inflammatory activation. 0.1-20 μM CKBA was added to LPS-activated BV2 and cultured for 24 hours. It was found that CKBA could significantly inhibit the proliferation of inflammatory microglia and maintain the stability of peroxisomes, thus exerting an anti-inflammatory effect ( Figure 4 ).

[0169] The Seahorse Energy Metabolism Assay calculates the OCR of cells by measuring the change in oxygen consumption reagents and the dissolved oxygen content in the medium surrounding the cells. Figure 4 , showing that CKBA can significantly inhibit the activity of inflammatory microglia.

[0170] Therefore, CKBA plays an important therapeutic role in inhibiting neuroinflammation by targeting MFE-2 to inhibit microglial inflammation.

[0171] 2. CKBA improves disease symptoms in AD animals

[0172] AD mice were intragastrically administered with CKBA ( Figure 5 A), conducted an in vivo intervention study, and continued the intervention for 3 months. The results showed that compared with the control group without CKBA, the number of platform crossings (platform crossing number) increased significantly, the time in the opposite quadrant (time in opposite quadrant) decreased significantly, and the time in the specific effective area (central area) as a percentage of the total exercise time (% of total time) increased significantly. Therefore, CKBA can significantly slow down the central higher functions such as learning and memory in AD mice ( Figure 5 B).

[0173] Therefore, CKBA has great value in potentially inhibiting central nervous system inflammation and slowing down disease progression in AD by targeting MFE-2.

[0174] The above results indicate that CKBA compounds can effectively improve the phenotype of central nervous system inflammation, and their therapeutic effect on central nervous system inflammation is unexpected.

[0175] Example 5: Synergistic effect of CKBA and donepezil on improving central nervous system diseases in animals

[0176] In order to obtain a substance that can be used in combination with CKBA to further enhance the effect, the inventors conducted a lot of research and screening work. The results showed that donepezil can be used in combination with CKBA to achieve a significant synergistic effect.

[0177] AD mice were given CKBA (100 mg / kg / day) and donepezil (3 mg / kg / day) by oral gavage for in vivo intervention study.

[0178] like Figure 6After 3 months of continuous intervention, it was shown that CKBA combined with donepezil could significantly slow down the degeneration of the learning, memory and other central higher functions of AD mice. There was a significant difference between the male mouse model group and the normal group, and there was a significant difference between the CKBA combined with donepezil group and the CKBA alone and donepezil alone model groups. It can be seen that the CKBA combined with donepezil group has a better improvement on the spatial exploration ability of mice.

[0179] The results of the water maze experiment showed that CKBA combined with donepezil had a further and better improvement on the learning and memory abilities of AD model animals.

[0180] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Use of the compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, for preparing a pharmaceutical composition for relieving or treating central nervous system inflammation; in, R is independently selected from the group consisting of hydrogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen.

2. The use according to claim 1, characterized in that The compound represented by formula (I) or its isomer, solvate or precursor, or pharmaceutically acceptable salt thereof targets and binds to hydroxysteroid 17-β dehydrogenase 4, thereby maintaining the structural stability or activity of hydroxysteroid 17-β dehydrogenase and the stability or activity of peroxisomes, thereby alleviating or treating central nervous system inflammation; or The compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, inhibit neuroinflammation associated with excessive activation of glial cells, alleviate the rate of neuronal damage or death, and improve the metabolic microenvironment of central nervous system cells by targeting hydroxysteroid 17-β dehydrogenase 4.

3. The use according to claim 1, characterized in that The central nervous system inflammation is central nervous system inflammation with dysfunction of hydroxysteroid 17-β dehydrogenase 4.

4. The use according to claim 1, characterized in that The central nervous system inflammation includes: central nervous system degenerative diseases or chronic central nervous system inflammation; preferably, The central nervous system degenerative diseases include: Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and learning ability or memory impairment.

5. Use of the compound represented by formula (I) or its isomer, solvate or precursor, or a pharmaceutically acceptable salt thereof, for preparing a pharmaceutical composition for maintaining the structural stability or activity of hydroxysteroid 17-β dehydrogenase 4, or maintaining the stability or activity of peroxisomes; in, R is independently selected from the group consisting of hydrogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen.

6. Use of the compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, for preparing a pharmaceutical composition for inhibiting neuroinflammation associated with excessive activation of glial cells, alleviating the rate of neuronal damage or death, or improving the metabolic microenvironment of central nervous system cells; in, R is independently selected from the group consisting of hydrogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen.

7. The use according to claim 1, 5 or 6, characterized in that The pharmaceutical composition further comprises donepezil; preferably, the ratio of the compound represented by formula (I) to donepezil is 10 to 60:1; more preferably 15 to 50:1; more preferably 20 to 45:

1.

8. The use according to claim 1, 5 or 6, characterized in that The compound represented by formula (I) has a structure represented by formula (II).

9. A composition for alleviating or treating central nervous system inflammation, comprising: A compound represented by formula (I) or its isomer, solvate or precursor, or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutical composition further comprises donepezil; preferably, the ratio of the compound represented by formula (I) to donepezil is 10 to 60:1; preferably 15 to 50:1; more preferably 20 to 45:

1.

10. A medicine box or test kit for alleviating or treating central nervous system inflammation, comprising the composition according to claim 9.

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