Application of SKI-178 in preparation of medicine for treating neurodegenerative diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of SKI-178 in preparation of medicines for treating or preventing neurodegenerative diseases related to abnormal sphingomyelin metabolism. The neurodegenerative disease related to abnormal metabolism of sphingomyelin is a disease related to down-regulation of expression or activity of Sgpp2; the neurodegenerative disease is accompanied by myelin sheath injury; the neurodegenerative diseases are selected from cognitive impairment related to hyperhomocysteinemia HHcy, Alzheimer's disease, Parkinson's disease, multiple sclerosis and vascular dementia; the SKI-178 plays a therapeutic role by improving metabolic disorder of sphingomyelin and / or repairing myelin sheath injury; the application is to up-regulate the expression of the homocysteinemia virulence gene Sgpp2 and / or enhance the activity of the homocysteinemia virulence gene Sgpp2. The invention finds that the SKI-178 small-molecule compound has potential application value in treatment of neurodegenerative diseases related to hyperhomocysteinemia, myelin sheath injury and Sgpp2 gene expression abnormality, and the SKI-178 small-molecule compound has potential application value in treatment of neurodegenerative diseases related to hyperhomocysteinemia, myelin sheath injury and Sgpp2 gene expression abnormality.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of SKI-178 in the preparation of a medicament for treating neurodegenerative diseases. Background Technology

[0002] Hyperhomocysteinemia (HHcy), a pathological condition caused by vitamin B12 / folic acid deficiency or inherited metabolic defects, drives neurodegenerative diseases, myelin damage, and sphingomyelin metabolism disorders through multiple interactive mechanisms: Elevated homocysteine ​​(Hcy) directly induces mitochondrial oxidative stress and calcium overload-induced apoptosis in neurons, promotes abnormal aggregation of β-amyloid (Aβ) and α-synuclein, and accelerates the neurodegenerative process of Alzheimer's disease, Parkinson's disease, and other diseases; simultaneously, Hcy damages vascular endothelial function, triggers cerebral small vessel ischemia, leading to oligodendrocyte death and white matter demyelination, and profoundly interferes with sphingomyelin metabolism—consuming methyl... The donor S-adenosylmethionine (SAM) inhibits the activity of key methyltransferases, hindering the conversion of ceramides to sphingomyelin and disrupting the stability of the myelin lipid bilayer. The abnormal accumulation of sphingomyelin degradation products (such as ceramides) further activates inflammasomes and apoptosis pathways, forming a vicious cycle of "demyelinating-sphingomyelin metabolic imbalance." Clinical studies have confirmed that the levels of sphingomyelin metabolites in the cerebrospinal fluid of HHcy patients are abnormal and significantly correlated with the severity of white matter lesions and cognitive decline. Although B vitamin supplementation can reduce Hcy levels, its reversal effect on existing sphingomyelin metabolic disorders and neurological structural damage is still limited, highlighting the necessity of early intervention in this multidimensional pathological network.

[0003] In neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis), sphingolipids, as key components of cell membranes and myelin sheaths, play a crucial role in maintaining the structural integrity of myelin and neurotransmission function through metabolic balance. When the activity of enzymes related to sphingolipid metabolism (such as sphingomyelinase and ceramidinase) is abnormal or there are genetic defects, it can lead to impaired synthesis, decomposition, or transport of sphingolipids. This results in the accumulation of intermediate products such as ceramides and sphingosine, or the loss of functional sphingolipids such as sphingomyelin and cerebrosides. Consequently, it can damage the proliferation and differentiation capacity of oligodendrocytes, leading to impaired myelin synthesis, decreased myelin stability, demyelinating lesions, or impaired myelin repair. Ultimately, this can cause slowed nerve axonal conduction velocity, disordered nerve signal transmission, and exacerbation of neuronal damage and disease progression. In existing technologies for treating myelin damage caused by sphingolipid metabolism disorders in neurodegenerative diseases, there are several obvious problems and drawbacks, which are precisely the problems that the present invention can solve: First, when exogenously supplementing sphingolipid precursors, the lack of specific targeting structures for oligodendrocytes or the central nervous system leads to their non-specific distribution throughout the body, which not only reduces the effective concentration acting on the target tissue, but also causes toxic side effects such as lipid metabolism disorders due to accumulation in organs such as the liver and kidneys; Second, in enzyme replacement therapy, recombinant enzymes have a half-life of only 4-6 hours due to their own molecular structure characteristics and degradation by proteases in the body, requiring multiple injections per week to maintain an effective concentration, causing great suffering and economic burden to patients, and repeated administration will activate the body's immune system to produce antibodies, neutralizing the activity of recombinant enzymes. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the background art by proposing the use of SKI-178 in the preparation of a medicament for treating neurodegenerative diseases.

[0005] The first aspect of the present invention provides the use of SKI-178 in the preparation of a medicament for treating or preventing neurodegenerative diseases associated with abnormal sphingomyelin metabolism.

[0006] Preferably, the neurodegenerative disease associated with abnormal sphingomyelin metabolism is a disease associated with downregulation of Sgpp2 expression or activity.

[0007] Preferably, the neurodegenerative disease is accompanied by myelin sheath damage.

[0008] Preferably, the neurodegenerative disease is selected from hyperhomocysteinemia (HHcy)-related cognitive impairment, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and vascular dementia.

[0009] Preferably, the SKI-178 exerts its therapeutic effect by improving sphingomyelin metabolism disorders and / or repairing myelin damage.

[0010] Preferably, the use is to upregulate the expression of the homocysteine-causing gene Sgpp2 and / or enhance its activity.

[0011] A second aspect of the present invention provides a pharmaceutical composition for treating or preventing the aforementioned diseases, comprising a therapeutically effective amount of SKI-178 and one or more pharmaceutically acceptable carriers or excipients.

[0012] Preferably, the dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

[0013] Compared with the prior art, the present invention has the following beneficial technical effects:

[0014] A novel mechanism based on the downregulation of Sgpp2 expression by high homocysteine ​​(HHcy) Figure 1 This invention is the first to screen and verify SKI-178 (chemical structure see below). Figure 3 As a small molecule compound targeting Sgpp2 ( Figure 2 Among 50 compounds, SKI-178 ranked second in activation effect on sphingolipid synthesis [Ratio = 3.321344061]. Simultaneously, it specifically binds to the Sgpp2 protein (molecular docking chemical energy S = -6.47743). Figure 4 Activating the sphingolipid synthesis pathway, the optimal concentration was determined to be 5 μM (MTT assay) based on cell viability testing. Figure 5 Furthermore, at this concentration, the content of sphingomyelin metabolites was significantly increased (LC-MS analysis showed an increase compared to the HHcy group). Figure 6 In the HHcy rat model, the SKI-178 treatment group (20 mg / kg / d orally for 4 weeks) showed a significantly increased MBP immunofluorescence intensity compared to the HHcy group (P<0.05). Figure 7 This indicates significant repair of the myelin sheath structure, and a significant improvement in the new object recognition index (P<0.05). Figure 8 The escape latency in the Morris water maze was shortened to (P<0.01). Figure 9 Furthermore, oral administration did not trigger an immunogenic response, highlighting its significant advantages in terms of therapeutic window, from in vitro (high efficacy at 5 μM) to in vivo (safety at 20 mg / kg). Attached Figure Description

[0015] Figure 1 High homocysteine ​​levels downregulate Sgpp2 expression.

[0016] Figure 2 Ranking of ratio curves for 50 drug compounds involved in the sphingolipid synthesis pathway.

[0017] Figure 3 The chemical structural formula for SKI-178 is given.

[0018] Figure 4 SKI-178 can bind to Sgpp2.

[0019] Figure 5 This is the optimal drug concentration for SKI-178.

[0020] Figure 6 SKI-178 promotes sphingomyelin synthesis in the sphingolipid synthesis pathway.

[0021] Figure 7 SKI-178 can improve myelin damage in HHcy rats.

[0022] Figure 8 SKI-178 can improve the cognitive ability of new things in HHcy rats.

[0023] Figure 9 SKI-178 can improve the spatial learning and memory abilities of HHcy rats. Detailed Implementation

[0024] Example 1

[0025] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0027] This invention provides the application of SKI-178 in the following products:

[0028] 1) Medications for treating neurodegenerative diseases;

[0029] 2) Medications for treating neurodegenerative diseases associated with hyperhomocysteinemia;

[0030] 3) Medications for treating neurodegenerative diseases related to myelin sheath injury;

[0031] 4) Drugs for treating neurodegenerative diseases caused by abnormal Sgpp2 gene expression leading to myelin damage.

[0032] The inventors of this invention discovered that HHcy SD rats exhibit significantly downregulated expression of the sphingomyelin synthesis gene Sgpp2. Through classic "novelty recognition test" and "water maze test," these HHcy rats demonstrated decreased learning and memory abilities. Pathological analysis revealed significant myelin damage in the HHcy rats. The inventors found that the small molecule compound SKI-178 can target Sgpp2 and improve sphingomyelin disorders, thereby effectively repairing myelin sheath damage and improving learning and memory deficiencies in HHcy rats. This finding suggests that the small molecule compound SKI-178 has potential application value in the treatment of neurodegenerative diseases associated with hyperhomocysteinemia, myelin damage, and abnormal Sgpp2 gene expression.

[0033] Example 1

[0034] HHcy levels downregulate Sgpp2 expression.

[0035] (1) HHcy cell model. OLN-93 cells were stimulated with 100 μl homocysteine ​​for 24 h. After 24 h, OLN-93 cells were lysed with RIPA lysis buffer or RNAisolater, and the changes in Sgpp2 mRNA and protein levels were detected by RT-qPCR and Western blotting.

[0036] (2) Experimental results. HHcy significantly downregulated the mRNA and protein levels of Sgpp2 (e.g., Figure 1 (As shown).

[0037] The specific experimental steps for RT-qPCR are as follows:

[0038] 1) RNA Extraction: Add an appropriate amount of RNAisolater Total RNA Extraction Reagent to the cell sample, ensuring complete coverage of the tissue block. Mix thoroughly by pipetting repeatedly and transfer to a 1.5 ml centrifuge tube. Incubate on ice for 5 minutes to promote lysis. Then add 1 / 5 volume of chloroform as lysis buffer, and vigorously vortex for 15 seconds until the solution emulsifies and turns milky white. Incubate at 4°C for 5 minutes. After centrifugation at 12,000×g for 15 minutes at 4°C, the solution will separate into three layers: a colorless aqueous phase containing RNA in the upper layer, a white protein layer in the middle layer, and a red organic phase in the lower layer. Carefully aspirate the upper aqueous phase into a new centrifuge tube, add an equal volume of pre-chilled isopropanol, gently invert to mix, and incubate at 4°C for 10 minutes. Centrifuge at 12,000×g at 4°C for 10 minutes; a white RNA precipitate will be visible at the bottom of the tube. Discard the supernatant, add 1 ml of RNase-free 75% ethanol (prepared with ddH2O), gently tap the bottom of the tube to resuspend the precipitate, and incubate at room temperature for 5 minutes. Centrifuge again at 12,000×g at 4℃ for 5 minutes to discard the ethanol. Remove the cap and allow the precipitate to dry at room temperature for 2-5 minutes (avoid over-drying). Finally, dissolve the precipitate with an appropriate amount of RNase-free ddH2O, gently pipetting to aid dissolution. Aliquot the resulting RNA solution and store at -80℃. Concentration and purity are determined using a spectrophotometer (A260 / A280 = 1.8-2.2; concentration is calculated as OD260 × dilution factor × genomic DNA removal and reverse transcription).

[0039] 2) Reverse transcription: Prepare a genomic DNA removal system in an RNase-free centrifuge tube: 12 μl RNase-free ddH2O, 4 μl 4×g DNAwiper Mix, and 1 pg–1 μg total RNA. Gently mix and incubate at 42°C for 2 minutes. Then, add 4 μl 5×HiScript III qRT SuperMix to the same reaction tube and gently mix to form a 20 μl reverse transcription system. Place in a PCR instrument and perform the following reaction program: reverse transcription at 37°C for 15 minutes, followed by inactivation of reverse transcriptase at 85°C for 5 seconds. The resulting cDNA can be used immediately for qPCR or stored at -20°C.

[0040] 3) qPCR amplification and detection: A 10 μl reaction mixture was prepared using the SYBR Green method: 5 μl 2×ChamQ SYBRqPCR Master Mix (ROX-free), 0.2 μl forward primer (10 μM), 0.2 μl reverse primer (10 μM), 1.0 μl cDNA template (diluted to an appropriate concentration), and 3.6 μl RNase-free ddH2O. The three-stage program was run in the qPCR instrument.

[0041] ① Pre-denaturation: 95℃ for 30 seconds (1 cycle);

[0042] ② Amplification: 95℃ for 10 seconds → 60℃ for 30 seconds (40 cycles);

[0043] ③ Melting curve: 95℃ for 15 seconds → 60℃ for 60 seconds → 95℃ for 15 seconds.

[0044] The specific steps of Western blotting are as follows:

[0045] 1) Add an appropriate amount of weak RIPA lysis buffer to the cell sample according to the tissue lysis buffer ratio (1:15) (add strong RIPA lysis buffer to the tissue), then sonicate on ice (amplitude 35%, 5-second pulse / 10-second interval, 5 cycles). After centrifuging the lysis buffer at 12,000×g 4℃ for 15 minutes, collect the supernatant and quantify the protein concentration using a BCA kit.

[0046] 2) After quantification, add an appropriate amount of loading buffer and boil at 95°C for 10 minutes, then cool to room temperature.

[0047] 3) Prepare a 10%-12% SDS-PAGE gel, load 20ug of boiled protein onto the gel, then concentrate the protein by electrophoresis at 80V for 30min, and finally separate the protein by electrophoresis at 120V for 45min.

[0048] 4) Transfer membrane: Soak filter paper, sponge, methanol-activated PVDF membrane, and separating gel in electrotransfer buffer. Then, clamp the membrane in the following order: white side of transfer clamp, sponge, filter paper, PVDF membrane, separating gel, filter paper, sponge, and black side of transfer clamp. Place the membrane in the transfer apparatus, pour in electrotransfer buffer, and run at a constant current of 200mA for 90 minutes in an ice bath.

[0049] 5) After the transfer is complete, remove the PVDF membrane and block it with 5% skim milk powder (prepared with 1×TBST) at room temperature for 1 hour. Then, wash the PVDF membrane slightly, cut the corresponding band according to the molecular weight of the target protein, and finally put the band into the diluted primary antibody solution (Sgpp2, β-actin) and incubate overnight at 4°C with shaking.

[0050] 6) After incubation with primary antibody, wash the bands three times with 1×TBST for 10 min each time, then add an appropriate amount of diluted secondary antibody (goat anti-mouse or goat anti-rabbit) and incubate on a shaker at room temperature for 1 h.

[0051] 7) After incubation with the secondary antibody, wash the bands three times with 1×PBST for 10 min each time. Then mix eECL-A and eECL-B (E41104, Vazyme) in equal volumes at a ratio of 1:1, and add them evenly to the target band in the dark. Expose and image the bands using a chemiluminescence imager.

[0052] Example 2

[0053] Screening for small molecule compounds that promote Sgpp2 activity and protein expression levels.

[0054] This example illustrates the existence of a small molecule compound, SKI-178, that can enhance the activity and protein expression level of Sgpp2. This drug screening method was based on a systematic screening and molecular validation of 50 candidate drugs, ultimately identifying SKI-178 as the ideal drug.

[0055] (1) Drug screening methods

[0056] Compound library construction: 50 candidate drug compounds were selected to construct a drug compound library.

[0057] Determination of optimal concentration: By consulting relevant literature, the optimal concentration data of each drug in OLN-93 cells were obtained to ensure that the administered concentration has a physiological effect and does not affect cell viability.

[0058] Cell model construction: OLN-93 cells were stimulated with 100 μl homocysteine ​​for 24 hours to serve as an experimental model in order to simulate the cellular environment of neurodegenerative diseases.

[0059] Drug treatment: In the cell model, each drug was treated uniformly at its optimal concentration. After adding each drug, the cells were cultured for another 24 hours.

[0060] Supernatant collection: 24 hours later, the cell culture supernatant of each experimental group was collected and stored at 4°C for subsequent testing.

[0061] (2) ELISA kit detection

[0062] The Sgpp2 content in the supernatant of each sample was quantitatively determined using an ELISA kit targeting Sgpp2. Based on the ELISA results, the ratio of Sgpp2 content in each drug group to the control group was calculated, and a ratio curve was plotted to show the effect of different drugs on Sgpp2 expression.

[0063] (3) Molecular docking analysis

[0064] For the screened candidate drugs, molecular docking analysis was performed to calculate the binding energy between the drug and Sgpp2, and to predict the potential mechanism of action of the drug on Sgpp2. Based on the ratio curve ranking and the activation energy data of molecular docking, the efficacy and affinity (e.g., ...) of each drug were comprehensively evaluated. Figure 2 (As shown).

[0065] (4) Determining the optimal drug

[0066] Among all candidate drugs, SKI-178 (molecular formula as follows) is the most promising. Figure 3As shown, it exhibits relatively optimal performance and has a high binding energy (e.g., Figure 4 (As shown).

[0067] Specifically, it exhibits a high Sgpp2 expression ratio in ELISA kit detection. The molecular docking activation energy is high, demonstrating good binding ability and potential.

[0068] (5) Determine the optimal cell drug concentration for SKI-178

[0069] MTT assay results showed that the inhibitory effect of SKI-178 on the proliferation of OLN-93 cells was significantly concentration-dependent (e.g., Figure 5 As shown in the figure, its half-maximal effective concentration (EC50) is approximately 5 μM. At low concentrations (e.g., 0-2 μM), SKI-178 did not significantly affect cell viability; when the concentration approached the EC50 value (10-50 μM range), cell viability decreased in a gradient manner; and when the concentration exceeded 10 μM, cytotoxicity significantly increased. Based on the concentration-effect curve, the optimal concentration of SKI-178 was determined to be 5 μM, which effectively inhibits cell proliferation while avoiding excessive killing, making it suitable for subsequent mechanism studies.

[0070] Example 3

[0071] SKI-178 can improve sphingomyelin synthesis in the sphingomyelin synthesis pathway of HHcy rats.

[0072] (1) Construction of the HHcy model rat: Eight-week-old healthy male Sprague-Dawley rats (purchased from Beijing Sprague Biotechnology Co., Ltd.) were used in this experiment. All animal operations strictly followed the experimental protocol approved by the Animal Ethics Committee of Jiangnan University. The rats were housed individually in a standardized laboratory environment, with free access to drinking water and standard rodent feed. The housing environment was set to a reverse light cycle of 12 hours of light / 12 hours of darkness (the light period was at night to meet the time requirements of subsequent behavioral tests). The experimental period lasted for 21 days. From 8:30 to 11:00 a.m. each day, the rats were injected sequentially via tail vein: the control group was injected with sterile saline, and the experimental group was injected with homocysteine ​​(L-Homocysteine, a product of Sigma-Aldrich, USA, with a daily dosage strictly calculated at 400 μl / kg body weight) dissolved in saline. SKI-178 treatment: 8-week-old HHcy rats were given SKI-178 via intraperitoneal injection (20 mg / kg / day) for one month. The injection was performed in the dark to maintain the stability of the compound, and the daily injection volume was kept constant after being calibrated by body weight.

[0073] During the last 7 days of the experiment (days 15 to 21), the spatial learning and memory abilities of rats were assessed using standardized paradigms such as the Morris water maze. Testing was conducted at the beginning of each day's dark cycle to match their active physiological rhythms. Within 24 hours of the behavioral tests, all rats were euthanized under deep anesthesia using ethically sound methods such as cardiac perfusion or cervical dislocation to ensure timely tissue collection for subsequent molecular biological or histological analysis.

[0074] (2) Experimental Results. Sphingomyelin and its metabolites were significantly decreased in HHcy rats. Neuronal levels of sphingomyelin and its metabolites were increased in HHcy rats after administration of SKI-178. Results are presented as mean ± SEM; statistical methods included two-way ANOVA (e.g., σ² ± ... Figure 6 (As shown).

[0075] The specific steps for non-targeted lipidomics testing are as follows:

[0076] 1) Sample preparation

[0077] Take 30 mg of tissue or 200 μl of biofluid, add a mixture of methyl tert-butyl ether / methanol / water (MTBE method, volume ratio 10:3:2.5), sonicate in an ice bath, and centrifuge to separate the phases. Collect the upper organic phase, dry it under nitrogen, redissolve it in isopropanol / acetonitrile (1:1), filter it through a 0.22 μm membrane, and store it at -80 °C.

[0078] 2) Chromatography-mass spectrometry analysis

[0079] UHPLC-Q-TOF platform was used:

[0080] Chromatography: C18 column (2.1×100mm, 1.7μm), elution with gradient of acetonitrile / water (containing 0.1% formic acid + 10mM ammonium formate) and isopropanol / acetonitrile (15min), column temperature 55℃;

[0081] Mass spectrometry: ESI positive / negative ion dual-mode scanning (m / z 100-1700), data-dependent acquisition (DDA) triggered secondary fragmentation.

[0082] 3) Quality control design:

[0083] A pooled quality control (QC) is inserted for every 10 samples; randomized injection sequences are interspersed with solvent blanks;

[0084] System suitability test: Standard lipid retention time offset <0.1 min.

[0085] 4) Data processing

[0086] The original data was peak aligned and normalized (QC-LOESS) to extract characteristic peaks;

[0087] Lipid identification: Level 1 precise quality (error <5ppm) + Level 2 fragment matching LIPID MAPS database.

[0088] Example 4

[0089] SKI-178 can improve myelin damage in HHcy rats.

[0090] Experimental Results. MBP density in the CA1 region of the hippocampus was significantly decreased in HHcy rats. After administration of SKI-178 to HHcy rats, the density of dendritic MBP increased in heterozygous mice. N=6 (each group consisted of 3 rats). Results are presented as mean ± SEM; statistical methods included Tow-way ANOVA (e.g., ...). Figure 7 (As shown).

[0091] The specific steps for immunofluorescence staining are as follows:

[0092] 1) Sample processing

[0093] The tissue was fixed with 4% paraformaldehyde for 24 hours (4℃) → dehydrated with 30% sucrose and settled → embedded with OCT → flash-frozen in liquid nitrogen and stored at -80℃.

[0094] 2) Slice preparation

[0095] Cut 8-12μm thin sections at -20℃, attach them to poly-L-lysine glass slides, and store at -80℃.

[0096] 3) Staining process

[0097] After rewarming, wash with PBS → permeabilize with 0.3% Triton X-100 for 15 min → block with 5% donkey serum for 1 h → incubate with primary antibody at 4°C overnight (e.g., anti-GFAP 1:500) → wash 3 times with PBS → incubate with fluorescent secondary antibody at room temperature in the dark for 1 h (e.g., Alexa Fluor 488, 1:500) → stain nuclei with DAPI for 5 min → mount with anti-quencher.

[0098] 4) Imaging analysis

[0099] Images were acquired using confocal microscopy (Z-stack scanning), and the fluorescence intensity was quantified using ImageJ.

[0100] Example 5

[0101] SKI-178 enhances the learning and memory abilities of HHcy rats.

[0102] 1) Novel Object Recognition Experiment. For three days prior to the formal experiment, rats were placed in a controlled-temperature, humidity, and lighting behavioral testing room daily for environmental acclimatization. During this period, each rat was placed individually in a clean white acrylic test box (60×60×40cm) and allowed to explore freely for 10 minutes. Simultaneously, their tails were marked with a non-toxic staining pen to eliminate environmental stress. The formal test consisted of two phases: During the training phase (Day 1), two identical objects (AA combination) were fixed diagonally across the box. The background was reset by replacing the bedding and rotating the box. The rat was then gently placed in the center of the box with its head facing away from the objects, and its exploration behavior was recorded for 10 minutes. During the testing phase (Day 2, 24-hour interval), one of the objects was replaced to form a new combination (AB). The bedding type was changed again, and the lighting angle was adjusted to reset the background. Behavior was recorded for 10 minutes in the same manner. Strict adherence to behavioral criteria was used: valid exploration was considered when the rat's nose was more than 1cm from the object and it actively sniffed; standing on the object with all four limbs was considered invalid. In the statistical analysis phase, the exploration time (tB) for the new object (B) and the exploration time (tA) for the old object (A) during the test period were extracted. One-way ANOVA was used for inter-group comparisons, and the results are presented as mean ± standard error (Mean ± SEM).

[0103] Depend on Figure 8 It can be seen that HHcy rats have a significantly reduced ability to recognize new things compared to the control group; while SKI-178 can significantly improve the ability of HHcy rats to recognize new things.

[0104] 2) Water Maze Experiment. One week prior to the experiment, rats were placed in a water maze laboratory (temperature 23±1℃, background noise ≤50 dB) to acclimatize. Formal training lasted 6 days, with 4 rounds of testing each day (round intervals ≥30 minutes). The water maze setup consisted of a circular pool with a diameter of 122 cm and a hidden platform fixed in the southeast quadrant (6 cm in diameter, platform top 1 cm below the water surface). The pool water was mixed with black ink to achieve a black, opaque state (constant water temperature 22-25℃). High-contrast geometric reference objects (circles, triangles, and squares with black patterns on a white background, fixed to the fence posts) were placed on three sides of the pool wall. During each round of testing, the rat was gently placed horizontally into the water facing the pool wall. An automatic tracking system recorded its platform-seeking latency (maximum 60 seconds): if it successfully climbed onto the platform within 60 seconds and remained there stably for 5 seconds, it was allowed to continue staying on the platform until a cumulative 20 seconds were reached before being removed; if it failed to climb the platform within the time limit, the operator guided it to the platform and forced it to stay there for 20 seconds. After each rat was removed from the water, its fur was immediately dried with a preheated towel, and it was placed back in a 38°C incubator. For the final analysis, the data from each group were integrated using the individual mean of 15 rats (6 days × 4 rounds = 24 latency periods per rat), and averaged within the group to form the daily data points. Figure 8The results are expressed as mean ± SEM of the daily latency (seconds), and the interaction effect of "treatment factor × training days" was evaluated using two-way ANOVA.

[0105] Depend on Figure 9 It can be seen that HHcy rats have significantly reduced spatial learning and memory abilities compared to control rats; while SKI-178 can significantly improve the spatial learning and memory abilities of HHcy rats.

[0106] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. Use of SKI-178 in the preparation of medicaments for the treatment or prevention of neurodegenerative diseases associated with abnormal sphingomyelin metabolism.

2. The use according to claim 1, characterized in that, The neurodegenerative diseases associated with abnormal sphingomyelin metabolism are those related to the downregulation of Sgpp2 expression or activity.

3. The use according to claim 1 or 2, characterized in that, The neurodegenerative disease is accompanied by myelin sheath damage.

4. The use according to claim 3, characterized in that, The neurodegenerative diseases mentioned are selected from hyperhomocysteinemia (HHcy)-related cognitive impairment, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and vascular dementia.

5. The use according to claim 3, characterized in that, The SKI-178 exerts its therapeutic effect by improving sphingomyelin metabolism disorders and / or repairing myelin damage.

6. The use according to claim 5, characterized in that, The intended use is to upregulate the expression of the hyperhomocysteinemia-causing gene Sgpp2 and / or enhance its activity.

7. A pharmaceutical composition for treating or preventing the disease as described in any one of claims 1-6, characterized in that, It contains a therapeutically effective amount of SKI-178 and one or more pharmaceutically acceptable carriers or excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

9. The pharmaceutical composition according to claim 7, characterized in that, The content of SKI-178 in the pharmaceutical composition is 0.1 μM-50 μM.

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