Use of pentacyclic triterpene compound targeting hydroxysteroid 17-β dehydrogenase 4

Abstract

Provided is a use of a pentacyclic triterpene compound targeting hydroxysteroid 17-β dehydrogenase 4. The pentacyclic triterpene compound can effectively relieve or treat central nervous diseases (inflammatory diseases), including degenerative diseases of the central nervous system or chronic central inflammatory diseases. The pentacyclic triterpene compound can maintain the structural stability or activity of hydroxysteroid 17-β dehydrogenase and maintain the stability or activity of peroxisome, thereby relieving or treating central nervous inflammation. The pentacyclic triterpene compound can target hydroxysteroid 17-β dehydrogenase 4, inhibit neuroglial cell overactivation related neuroinflammation, relieve neuron damage or death rate, and improve the cell metabolism microenvironment of the central nervous system.

Description

Application of pentacyclic triterpenoid compounds targeting hydroxysteroid 17-β dehydrogenase 4

[0001] This application claims priority to patent application No. CN 202311510213.4, filed on November 13, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of medicine, and in particular relates to the application of a class of pentacyclic triterpenoid compounds targeting hydroxysteroid 17-beta dehydrogenase 4. Background Art

[0003] 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 the ability to take care of themselves and even die. It is considered 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. Given the mismatch between the growing market demand for AD and the production capacity of clinical drugs, and the current situation where 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.

[0004] Lewy bodies (LBs) are common in several degenerative central nervous system disorders, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). These LBs are rich in aggregated forms of α-synuclein (α-syn). α-syn is a 14-kDa protein with no well-defined structure, primarily produced in neurons. Under pathological conditions, the monomeric form of the protein gradually forms oligomers and insoluble fibrillar assemblies, accumulating intracellularly as LBs. 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 spread from cell to cell, contributing to disease progression. In microglia, α-syn and mitochondria can be transported and degraded via intercellular membrane protrusions, potentially contributing to this intercellular spread.

[0005] Multiple sclerosis (MS) is an incurable inflammatory autoimmune disease of the central nervous system (CNS) that affects millions of people worldwide. MS is a chronic demyelinating disease of the CNS whose onset and progression are driven by a combination of immune dysregulation, genetic predisposition, and environmental factors. Activation of microglia and astrocytes is a key player in MS immunopathology, playing specific roles at the anatomical site and stage of the disease and controlling demyelination and neurodegeneration. Laura A. Pasquini et al. suggest that the impaired phagocytosis of myelin debris following microglial depletion reflects the central role of microglia in demyelination. Insufficient phagocytosis subsequently impedes myelin regeneration, particularly in myelin-rich regions, and contributes to neurodegeneration. Astrocytes also participate in myelin resorption, particularly as an early response to injury that ultimately triggers immune cell recruitment. This early response can positively or negatively influence lesion pathology, depending on the inflammatory milieu, which is itself altered by microglial depletion. Overall, microglial depletion may disrupt the inflammatory landscape of demyelinating lesions, promoting either beneficial or detrimental responses of astrocytes and oligodendrocytes, thereby influencing neurodegeneration. Therefore, this study highlights the importance of regulating glial-mediated neuroinflammation as a drug target for relapsing-remitting MS, and suggests that drugs that regulate and maintain glial homeostasis have great potential in treating diseases such as multiple sclerosis.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide the preparation and application of a pentacyclic triterpene compound targeting hydroxysteroid 17-β dehydrogenase 4.

[0008] In a first 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 alleviating or treating central nervous system inflammation;

[0009] Wherein, R is independently selected from the group consisting of hydrogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, and halogen.

[0010] In one or more embodiments, 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.

[0011] In one or more embodiments, the compound represented by formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, inhibits neuroinflammation associated with excessive activation of glial cells (including microglia), alleviates the rate of neuronal damage or death, and improves the metabolic microenvironment of central nervous system cells by targeting hydroxysteroid 17-β dehydrogenase 4.

[0012] In one or more embodiments, the central nervous system inflammation is central nervous system inflammation caused by dysfunction of hydroxysteroid 17-β dehydrogenase 4 (including decreased or missing expression, missing or decreased activity, decreased stability, etc.).

[0013] In one or more embodiments, the central nervous system inflammation includes: central nervous system degenerative disease or chronic central nervous system inflammation.

[0014] 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 or memory impairment.

[0015] In another aspect of the present invention, provided is 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.

[0016] 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 central nervous system cells.

[0017] 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).

[0018] In one or more embodiments, the compound represented by formula (I) has a structure represented by formula (II).

[0019] The English name of the compound of formula (II) is 3-o-α-cyclohexanoyl-11-keto-β-boswellic acid, 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 a pharmaceutically acceptable salt thereof 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 of formula (I) or its isomer, solvate or precursor, or its pharmaceutically acceptable salt is used as the sole active ingredient (active component) in the composition (including pharmaceutical composition). Alternatively, the compound of formula (I) or its isomer, solvate or precursor, or its pharmaceutically acceptable salt, and donepezil are used as the sole 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 of which exerts an independent effect.

[0024] In one or more embodiments, the compound of formula (I) is prepared by the following method: using 11-carbonyl-β-acetylboswellic acid as a raw material, replacing the 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 weight volume ratio to suspend the powder to a final concentration of 10-20 mg / ml, and orally administering the compound 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 carboxymethylcellulose, 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, compressing the powder into large tablets using a single-punch tablet press, crushing the large tablets into granules using a mortar, and finally adding the lubricant boracic acid magnesium stearate to the granules and sieving them (such as a 30-mesh sieve) to form the granules, and directly compressing the granules using a single-punch tablet press to prepare 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 content and ratio level of the compound 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, 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 for AD; Conditional knockout of MFE-2 in microglia of AD mice (MFE-2 cKO 5xFAD mice, upper figure) were subjected to behavioral experiments (middle figure), Aβ and microglial cell staining, and immunofluorescence staining was used to determine that in the early stage of AD mice with MFE-2 conditional knockout, microglial cell morphology 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), suggesting that MFE-2 deficiency caused immune inflammatory dysfunction and aggravated the deposition of Aβ plaques in brain tissue.

[0037] Figure 2. CKBA compounds efficiently penetrate the blood-brain barrier after oral administration. Mass spectrometry was used to evaluate the blood-brain barrier penetrability of CKBA. Normal mice were gavage-administered 2.4 mg / mouse (100 mg / kg) of CKBA. One hour after gavage, fresh brain tissue and serum were collected for mass spectrometry analysis, demonstrating that CKBA effectively penetrates the blood-brain barrier.

[0038] Figure 3. CKBA compounds bind to the MFE-2 protein with high affinity. Studies on the anti-inflammatory effects of CKBA and its pharmacodynamic mechanism of action revealed that CKBA has a very strong affinity for MFE-2.

[0039] Figure 4. CKBA inhibits microglial inflammatory activation. In vitro cell experiments explored the effect of CKBA on microglial inflammatory activation. CKBA was added to LPS-activated BV2 cells and cultured for 24 hours. CKBA significantly inhibited the proliferation of inflammatory microglia, maintained the structural stability of MFE-2, inhibited protein degradation, and maintained peroxisome stability, exerting an anti-inflammatory effect (Panel A). Panel B shows a quantitative analysis of intracellular mitochondrial stress levels.

[0040] Figure 5. CKBA treatment effectively alleviates AD in animal models. In vivo intervention studies were conducted by gavage with CKBA in AD mice for 3 months.

[0041] Figure 6. The combined effect of CKBA and donepezil treatment was significantly superior to that of CKBA or donepezil alone. AD mice were treated with CKBA and donepezil orally for three months to assess the degree of deterioration in central nervous system functions, such as learning and memory. A water maze test was used to measure escape latency and platform crossovers, while a Y maze was used to assess spatial memory and learning abilities. DETAILED DESCRIPTION

[0042] The inventors have been dedicated to exploring new molecular therapeutic targets for the pentacyclic triterpenoid compounds of formula (I). After in-depth research and analysis, they have screened and identified a high-affinity functional target for the pentacyclic triterpenoid compounds of formula (I), namely, hydroxysteroid 17-β dehydrogenase 4 (MFE-2). Furthermore, the inventors' analysis has shown that the pentacyclic triterpenoid compounds of formula (I) can effectively alleviate or treat central nervous system diseases (inflammatory diseases), including: central nervous system degenerative diseases or chronic central nervous system inflammatory diseases.

[0043] the term

[0044] Those skilled in the art will understand that the term "alkyl" as 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 of is well known to those skilled in the art, and indicates that the group R can be substituted at any one or more substitutable positions on the ring. Furthermore, the choice 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, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, with a reasonable benefit / risk ratio.

[0053] In the present invention, a "pharmaceutically acceptable carrier" is a pharmaceutically or food-acceptable solvent, suspending agent, or excipient used to deliver the compound of formula (I), isomer, solvate, precursor, or pharmaceutically acceptable salt thereof to an animal or human. The carrier may be a liquid or a 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 catalyzes 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 contribute to the severe central nervous system dysfunction seen in D-bifunctional protein deficiency (DBPD).

[0057] Microglia are the most abundant immune cell type in the central nervous system, and the neuroinflammation they mediate 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 the microglia of 5xFAD mice, the brain showed abnormal microglial morphology, decreased immune surveillance function and persistent high activation phenotype at an earlier age compared to 5xFAD mice, and behavioral functions such as memory were significantly reduced. After intervention with pentacyclic triterpenoid compounds (preferably CKBA), the level of MFE-2 protein 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 function 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 a decrease in MFE-2 protein content can lead to severe central nervous system disorders, especially when the loss of MFE-2 in microglia leads to a 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 MFE-2 expression, ultimately alleviating the progression of neurodegenerative diseases. Therefore, CKBA inhibits central inflammation by targeting microglial cell 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 systematically to individuals in need, and is particularly capable of efficiently penetrating the blood-brain barrier, and then targeting the intracellular MFE-2 protein to maintain its normal intracellular metabolic function, stabilize the metabolic microenvironment of glial cells, inhibit overactivation, 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 triterpenoid 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 system inflammation related to persistent activation of microglial cells.

[0062] In preferred embodiments, "central nervous system inflammation" refers to a chronic, persistent, hyperinflammatory state within the central nervous system mediated by microglial activation, as well as other mechanisms of microglial-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 rapidly 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 compounds of formula (I), or their isomers, solvates, precursors, or pharmaceutically acceptable salts thereof, as long as they also have the same or substantially the same function as the compounds of formula (I). The "pharmaceutically acceptable salts" are salts formed by the reaction of the compounds with inorganic acids, organic acids, alkali metals or alkaline earth metals. 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 compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.

[0065] The "precursor of the compound" refers to a compound that undergoes metabolism or chemical reactions in the patient's body after being taken by an appropriate method and is converted into a compound of structural formula (I), or a salt or solution composed of a compound of chemical structural formula (I).

[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, abbreviated as CKBA. Its molecular formula C 37 H 56 O5, molecular weight 581.

[0067] Those skilled in the art will 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-acetylboswellic acid as a raw material, replacing the AcO- group thereof with Group. More preferably, the preparation steps include: (i) reacting 11-carbonyl-β-acetylboswellic acid (AKBA) 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 represented by formula (I). Other methods for preparing the compound represented by formula (I) are also included in the present invention. For example, 11-carbonyl-β-boswellic acid (KBA) can be used as a raw material and reacted with cyclohexanecarbonyl chloride to directly obtain the compound represented by 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; and (2) suspending the pentacyclic triterpene compound suspension in a 0.1-1% sodium carboxymethyl cellulose solution by weight to prepare a powdered pentacyclic triterpene compound suspension to a final concentration of 10-20 mg / ml. Preferably, 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%, to a final concentration of 10-20 mg / ml, and the tablet is orally administered at a drug weight ratio of 100±50 mg / kg; (2) the pentacyclic triterpene compound tablet is prepared by selecting three diluents: microcrystalline cellulose 101, carboxypropyl methylcellulose, and starch; and selecting three disintegrants: 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 dissolution rate of the drug; (3) the prepared pentacyclic triterpene compound powder is uniformly mixed with the diluent and disintegrant, compressed into large tablets using a single punch tablet press, and then the large tablets are crushed in a mortar to form granules, and finally the lubricant boaoko magnesium stearate is added to the granules and sieved through a 30-mesh sieve, and directly compressed into tablets using a single punch tablet press to prepare 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 triterpenoid 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 adjustable 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 compound is administered at a dosage of 50-150 mg / kg in vivo.

[0075] In a preferred embodiment of the present invention, the compound is administered into the body of a subject in need thereof by oral gavage / 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 cells to be treated.

[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. The individual dosage is 50-150 mg / kg / day.

[0080] In a preferred embodiment of the present invention, the pentacyclic triterpenoid compound is formulated into a diet / feed and administered with meals. It should be understood that the physician may also make appropriate dosage changes based on actual clinical conditions.

[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 medicated feed and / or oral tablets.

[0082] In a preferred embodiment of the present invention, a preferred highly lipophilic small molecule compound is provided, which is more preferably capable of effectively penetrating the brain barrier and being 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 disease includes any age-related degenerative disease of the central nervous system such as AD, MS, PD, etc., accompanied by a long-term and persistent activation of the 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 unlikely to recur 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, with a molecular weight of 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 covalent or non-covalent. 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 the form of a covalent bond, while some non-covalent connections (not forming a covalent bond), such as coupling, adsorption, binding, 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 therapy

[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] During administration, the pentacyclic triterpenoid compound can be administered first, followed by donepezil; or the order can be reversed; or the two can be administered simultaneously. It should be understood that various modes of administration are encompassed by the present invention.

[0098] The invention provides a mixture of small molecule compounds, comprising: 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 compositions or mixtures of the present invention can be prepared into any conventional dosage form by conventional methods. The dosage form can be varied, as long as it allows the active ingredient to effectively reach the mammalian body. For example, it can be selected from the group consisting of injections, infusions, tablets, capsules, and pills. 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 container suitable for injection or infusion. 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 remainder can be a pharmaceutically acceptable carrier.

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

[0103] The present invention also provides a medicine kit for alleviating or treating central nervous system inflammation, wherein the medicine kit 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 will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 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] CKBA compounds or their derivatives are used for systemic administration. CKBA (molecular formula: C37H56O5, molecular weight: 580.85, solubility: fat-soluble, soluble in methanol / DMSO, insoluble in water, independently synthesized). Cyclic boswellic acid (CKBA) is a small molecule compound obtained by structural modification and optimization of the active natural product molecule AKBA as a lead compound. The specific synthesis involves using AKBA as the starting material, hydrolyzing it under alkaline conditions to obtain the KBA intermediate, and then cyclohexanecarboxylating and purifying KBA to obtain the CKBA compound. The detailed synthetic route is as follows:

[0111] In vivo administration: Prepare a suspension using a 0.1-1% volume-to-mass ratio of sodium carboxymethylcellulose solution at a drug concentration of 15 mg / mL. Administer via oral gavage at a dose of 50-150 mg / kg / day. Preferably, administer CKBA with food or feed at a dose of 50-150 mg / kg / day.

[0112] Cell treatment: CKBA was dissolved in DMSO to a storage concentration of 5 mM (1000X) and stored at -20°C. The storage solution was added to the cell culture medium at a ratio of 1:1000 and mixed thoroughly to a final working concentration of 5 μM. Treat for 24-48 hours, then harvest the cells for subsequent experiments.

[0113] 2. Build MFE-2 cKO 5xFAD, 5xFAD, MFE-2 cKO 、MFE-2 cKI 5xFAD mice to investigate the regulatory effect of MFE-2 on microglial function

[0114] 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).

[0115] Cx3cr1CreHsd17b4 was obtained by breeding Hsd17b4fl / fl with Cx3cr1Cre and 5xFAD mice (Alzheimer's disease mice). fl / fl 5xFAD(MFE-2 cKO 5xFAD).

[0116] Take Hsd17b4 from the same litter fl / fl control) and Hsd17b4 fl / fl 5xFAD 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 CRISPR. fl / fl MFE-2 was obtained by breeding with Cx3cr1Cre and 5xFAD mice. cKI 、MFE-2 cKI 5xFAD).

[0117] The regulatory effect of MFE-2 on Microglia homeostasis and inflammatory activation was observed in mice at different stages.

[0118] 3. Immunofluorescence imaging analysis of microglia-mediated central nervous system inflammation in different animal models

[0119] According to the progression of central nervous system inflammation and the distribution characteristics of microglia, MFE-2 was injected into the glioma tissue at the early stage (2 months), the middle stage (4 months), and the late stage (8 months). cKO 5xFAD, 5xFAD, MFE-2 cKO The control mice were euthanized, and the blood in the brain tissue was completely removed by cardiac perfusion. After replacing the fixative with tissue fixative for systemic perfusion fixation, the intact brain tissue was collected and placed in the fixative for 24 hours. The tissue was then washed with PBS and dehydrated with gradient sucrose solution. The tissue was embedded in OCT and 30 μm thick tissue sections were prepared for immunofluorescence staining to analyze the expression levels of MFE-2, Iba-1, Cx3cr1, TNFα, F4 / 80, and IL-6.

[0120] Immunofluorescence staining procedures included incubation with 10% goat serum blocking solution, primary and secondary antibody incubation, and DAPI nuclear staining. After sealing, microglial distribution and morphological characteristics were imaged using a Leica SP8 laser scanning confocal microscope. Microglial morphology and intracellular fluorescence signal intensity were quantitatively analyzed using ImageJ software.

[0121] 4. Animal experiments and mass spectrometry methods on CKBA penetrating the blood-brain barrier

[0122] CKBA absolute quantitative detection experiment mass spectrometry standard solution and standard curve: CKBA standard methanol dilution (10 ng / mL, 1 ng / mL), preparation of standard curve: CKBA standard solutions with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 ng / mL were prepared in methanol. After centrifugation of the above solutions (18800g, 15 min, 4°C), 50 μL of the supernatant was transferred to a sample vial for mass spectrometry analysis; preparation of spiked blank serum standard curve (spiked standard): 10 μL of each concentration of CKBA working solution in the above CKBA standard methanol dilution was taken into 90 μL of blank serum and vortexed for 30 s.

[0123] 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℃) to take the supernatant and centrifuge again for use; Spiked blank brain tissue homogenate standard curve (Spiked Preparation of the CKBA standard: 10 μL of each CKBA working solution in the methanol dilution of the above CKBA standard was added to 90 μL of the blank brain tissue homogenate supernatant and vortexed for 30 seconds. Processing of the blank brain tissue homogenate sample: 50 μL of the blank brain tissue homogenate supernatant was placed in a sample vial for mass spectrometry analysis. Processing of the measured brain tissue homogenate sample: 50 μL of the measured brain tissue homogenate supernatant was placed in a sample vial for mass spectrometry analysis.

[0124] Detection method and process: The primary and secondary mass spectrometric information and corresponding mass spectrometric parameters of CKBA were determined using a 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 spectrometric 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, specified parent ion, the parent ion obtained from the primary mass spectrometry results was 581.6, and 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.

[0125] 5. Pull-down and Western blot experiments of CKBA-MFE-2 direct binding affinity

[0126] CKBA pull-dwon assay detects the direct interaction between CKBA and MFE-2. Specifically, the candidate small molecule drug CKBA was biotin-labeled, and CKBA-biotin was added to the culture medium (final concentration 5 μM) for 6 hours. The cells were washed and lysed, and then incubated with Streptavidin magnetic beads at 4°C for 3 hours with rotation. After washing the beads four times, the beads were added with loading buffer and boiled at 96°C for 5 minutes. Western blot (WB) was used to detect MFE-2 protein levels to evaluate the direct interaction between the small molecule compound and MFE-2.

[0127] The Western blot protein immunoblotting detection steps include: immersing fresh cells and tissue samples in lysis buffer, placing them on ice and letting them stand at low temperature for 10-15 minutes. After observing that the cells and tissues are fully lysed into a homogeneous turbid liquid, placing them in an ice bath again and letting them stand for 10 minutes. Centrifugation is carried out at 12000 rpm for 10 minutes, and the supernatant is collected. After the total amount of protein is detected using the BCA method, the steps of sample loading, electrophoresis, membrane transfer, blocking, primary antibody and secondary antibody incubation are carried out. After development with ECL luminescent developer, the protein content value is analyzed using ImageJ.

[0128] 6. CKBA treatment of LPS activated BV2 for CCK-8 experiment

[0129] Mouse microglial cell line BV2 was inoculated into 96-well flat-bottom plates and cultured overnight to allow cells to fully adhere. BV2 was stimulated and activated with LPS. LPS (Sigma) stock solution (5 mg / mL) was added to the supernatant of BV2 cell culture medium and mixed evenly. Cell morphology was observed after activation for 12 hours. CKBA working solution was further prepared according to the above method. CKBA was added to the cell culture medium at a final concentration of 5 μM and cultured for 6 to 24 hours.

[0130] Then, CCK8 (Japan Tongren) was used to detect cell viability. The cell culture medium of the 96-well plate was removed, and 100 μL of the diluted CCK8 reaction solution was added to the well plate. 100 μL was protected from light. Incubation conditions: 37°C incubator, about 1-2 hours. After 1 hour of incubation, the cells were removed and the cell color (orange) signal was detected using a multifunctional microplate reader (Tecan Infinite 200 Pro, Austria). Depending on the signal intensity, the cells could be removed at 1.5 / 2 hours of incubation, and the changes in the cell color (orange) signal were detected at multiple points. The color intensity value was recorded, and SPSS was used for statistical analysis and graphing.

[0131] 7. Seahorse experiment of CKBA treatment of LPS-stimulated BV2

[0132] The specific methods of CKBA treatment and LPS activation of BV2 cell lines are as described above.

[0133] The main steps of the Seahorse experiment include: Seahorse XF cell mitochondrial stress analysis: Use the above experimental method to obtain single cells from brain tissue, collect microglia in the brain tissue by flow cytometry, and seed primary microglia into Seahorse XF cell culture plates 4 to 7 days in advance. After complete attachment, hydrate the probe plate with double-distilled water one day in advance and place it in a CO2-free 37°C incubator 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 and add the corresponding substrates such as glucose, pyruvate, and glutamine in the kit to adjust the pH. 7.4. Wash the cells three times with the detection solution. Replace the cell culture medium with the detection solution and place in a CO2-free 37°C incubator within 1 hour before testing. Prepare the test drugs Oligo, FCCP, and ROT / AA and add them to the probe plate dosing wells A, B, and C. Then, load the tester and run the probe plate hydration plate and calibration process. Then, remove the hydration plate and replace the probe plate, and 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 based on the cell number, quantify the level of intracellular mitochondrial stress.

[0134] 8. CKBA oral preparation drug treatment plan

[0135] Administer by gavage and / or with feed and / or as oral tablets.

[0136] In an animal model, a 15 mg / mL CKBA suspension (solvent: 0.5% sodium carboxymethylcellulose) was administered orally with a volume of 200 μl (equivalent to a dose of 100 mg / kg) for 5 consecutive months, once every other day. The therapeutic effect was evaluated after the end of the administration period.

[0137] 9. Mouse models of neuroinflammation and degenerative diseases

[0138] (1) 5xFAD transgenic mice 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 neuron-specific regulatory element Thy1 promoter to drive transgene overexpression in the brain. This strain of mice has 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.

[0139] (2) Preparation of MS mouse (EAE) model and evaluation of CKBA intervention effect

[0140] Ten-week-old C57BL / 6 female mice were immunized with MOG35-55 emulsion and administered with PTX on the day of immunization and the next day. Twelve days after immunization, different doses of the small molecule drug FenCKBA were orally administered. The percentage change in body weight and clinical manifestations of each group were recorded. MOG35-55: myelin oligodendrocyte glycoprotein polypeptides 35 and 55. PTX: pertussis toxin. Fingolimod: fingolimod.

[0141] (3) Preparation of PD mouse model and evaluation of CBKA intervention effect

[0142] Healthy, male, SPF-qualified Balb / C mice, 4-6 weeks old and weighing 18-20 g, were intraperitoneally injected with MPTP at a dose of 20 mg / kg / day for 14 consecutive days. The control group received an equal volume of saline, using the same procedures and precautions. The model was successfully established after MPTP administration. Following treatment with the corresponding CKBA or control solvent, the mice were treated. The normal and PD model groups received an equal volume of PBS, using the same procedures and precautions.

[0143] (4) Evaluation of therapeutic effects in mouse models, behavioral experiments (rotarod, water maze, open field)

[0144] Evaluation of the therapeutic effect of CKBA on AD: 8-10 month old 5xFAD mice, 5xFAD mice treated with the small molecule drug CKBA, and control mice were selected for behavioral experiments including water maze, open field, and rotarod to evaluate the higher functions of the central nervous system. The behavioral experiments were fully recorded and analyzed offline using an animal motion trajectory tracking system (EthoVison XT 16.0).

[0145] Morris water maze: Mice were trained for 5 days of adaptive training, abnormal individuals were excluded, and a probe test was performed on the last day after the platform was removed. Quantitative data such as the platform latency after entering the water in each quadrant during the learning period, the number of platform crossings in the last trial, and the latency to enter the platform area were recorded.

[0146] Open field test: The spontaneous activity paths of mice in each area of ​​a 50cm*50cm*40cm unfamiliar open field were recorded for 5 minutes, and the movement distance and time of mice in the central area were analyzed to assess the anxiety changes of mice.

[0147] Rotarod test: The mice were subjected to three adaptive training sessions, followed by 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.

[0148] Example 1: Effect of MFE-2 knockout on pathological plaque deposition in the brains of AD animals

[0149] The inventors conditionally knocked out MFE-2 in microglia of AD mice (MFE-2 cKO 5xFAD mice; referred to as ADcKO) (Fig. 1, upper panel) were analyzed.

[0150] Comparison of 5xFAD and MFE-2 through behavioral experiments cKO Differences in higher-order central nervous system activity between the two groups of 5xFAD mice. As shown in the middle panel of Figure 1, the learning and memory abilities of the knockout group mice were significantly reduced compared to the control AD ​​mice, and their average anxiety levels were significantly increased.

[0151] Further staining of Aβ and microglia revealed that in the early stages of AD mice with conditional knockout of MFE-2, the morphology of microglia showed obvious abnormalities, 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, suggesting that MFE-2 deficiency caused immune inflammatory dysfunction and aggravated the deposition of Aβ plaques in the brain tissue (Figure 1, bottom).

[0152] 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.

[0153] 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.

[0154] Example 2: Penetration of CKBA through the blood-brain barrier

[0155] Whether CKBA can effectively alleviate AD is an exploration with great potential clinical value. The inventors used mass spectrometry to evaluate the blood-brain barrier penetration of CKBA. First, an effective method for absolute quantification of CKBA by mass spectrometry was established. The CKBA standard was detected using the optimized chromatography-mass spectrometry method described above. A 10 ng / mL CKBA sample was first injected, and a high characteristic chromatographic peak was observed at around 4.52 min. The concentration of the CKBA standard was reduced (1 ng / mL) and the injection analysis continued. A characteristic chromatographic peak was still observed at around 4.52 min, and its response was approximately 1 / 10 of that of the 10 ng / mL CKBA. The standard curve results showed good linearity and detection accuracy, indicating that the established quantitative method can accurately quantify the concentration of CKBA in methanol solution. The primary mass spectrometry scan yielded a parent ion of 581.6, indicating that CKBA had an additional proton in the mass spectrum. The secondary mass spectrometry scan yielded the corresponding product ion fragmentation spectrum, selecting the same quantification product ion, 407.5, as previously used by the company. Thus, the quantitative ion pair for CKBA was 581.6 / 407.5 (Figure 2A). Figure 2B demonstrates that the established CKBA quantification method is specific, and the standard curve exhibits good linearity.

[0156] The absolute content of CKBA in brain tissue was further detected based on the above-mentioned optimized method. For the detection of the absolute content of CKBA in brain tissue, normal mice were used. CKBA was administered orally 1 hour later, and then fresh brain tissue and serum were perfused and collected for mass spectrometry detection.

[0157] The results showed that CKBA could effectively penetrate the blood-brain barrier ( Figure 2C-E ).

[0158] Therefore, CKBA can effectively cross the blood-brain barrier, which is an important basis for the systemic use of CKBA to treat neurodegenerative diseases.

[0159] Example 3: CKBA directly binds to MFE-2 in living cells, protecting MFE-2's intracellular stability

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

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

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

[0163] The authors further explored the effect of CKBA on the MFE-2 protein. They treated normal BV2 cells with CKBA and found, by detecting protein expression, that CKBA did not affect baseline MFE-2 expression in the cells. LPS stimulation significantly downregulated MFE-2 expression in BV2 cells. CKBA treatment was able to effectively maintain MFE-2 expression and levels in the cells (Figures 3D-E).

[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 neurodegeneration in AD mice and exerts a therapeutic effect

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

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

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

[0169] The Seahorse energy metabolism assay measures the change in dissolved oxygen content between oxygen-consuming reagents and the culture medium surrounding the cells and calculates the OCR of the cells. As shown in Figure 4B, 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] An in vivo intervention study was conducted on AD mice gavage-administered CKBA (Figure 5A) for three months. Results showed that compared to a control group without CKBA, CKBA treatment significantly increased the number of platform crossings, decreased the time in the opposite quadrant, and significantly increased the percentage of total exercise time spent in the specific active zone (central zone). Therefore, CKBA significantly slowed down higher-level central functions such as learning and memory in AD mice (Figures 5B-C).

[0173] Therefore, CKBA has great potential to inhibit central nervous system inflammation and slow down the progression of 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 surprising.

[0175] Example 5: Synergistic Effects of CKBA and Donepezil on Improving Animal Central Nervous System Diseases

[0176] To obtain a substance that can be combined with CKBA to further enhance its efficacy, the inventors conducted extensive research and screening, and found 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 an in vivo intervention study.

[0178] As shown in Figure 6, after three months of continuous intervention, the combined use of CKBA and donepezil significantly slowed the deterioration of higher-level central nervous system functions, such as learning and memory, in AD mice. Significant differences were observed between the male model group and the normal control group. Significant differences were also observed between the CKBA and donepezil groups and the CKBA and donepezil alone groups. This suggests that the CKBA and donepezil combination significantly improved 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 effect on the learning and memory abilities of AD model animals.

[0180] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

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.

5. The use according to claim 4, characterized in that 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.

6. 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.

7. 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.

8. The use according to claim 1, 6 or 7, characterized in that The pharmaceutical composition also includes donepezil.

9. The use according to claim 8, characterized in that The ratio of the compound represented by formula (I) to donepezil is 10 to 60:

1.

10. The use according to claim 9, characterized in that The ratio of the compound represented by formula (I) to donepezil is 15 to 50:

1.

11. The use according to claim 10, characterized in that The ratio of the compound represented by formula (I) to donepezil is 20-45:

1.

12. The use according to claim 1, 6 or 7, characterized in that The compound represented by formula (I) is a compound having a structure represented by formula (II):

13. 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; The pharmaceutical composition also includes donepezil; the ratio of the compound represented by formula (I) to donepezil is 10 to 60:

1.

14. The composition according to claim 13, characterized in that The ratio of the compound represented by formula (I) to donepezil is 15 to 50:

1.

15. A medicine kit for alleviating or treating central nervous system inflammation, comprising the composition according to claim 13 or 14.

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