Application of GW9662 in treatment of meningeal carcinoma

Abstract

The invention discloses an application of GW9662 in treatment of meningeal carcinoma. The application finds that GW9662 can effectively inhibit meningeal carcinoma cells for the first time, the discovery of the application is expected to overcome the existing treatment difficulty, opens up a new path for the treatment of meningeal carcinoma, remarkably improves the treatment effect, prolongs the life of a patient, and has a wide application prospect clinically.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of GW9662 in the treatment of meningeal carcinoma. Background Technology

[0002] Meningeal carcinomatosis (MC), also known as leptomeningeal metastasis (LM), refers to the metastasis of malignant tumor cells, diffusely or focally infiltrating the pia mater and subarachnoid space. It is a special type of metastatic cancer of the central nervous system. Clinically, it mainly manifests as multifocal neurological dysfunction, such as symptoms of damage to the brain, cranial nerves, and / or spinal nerves.

[0003] Currently, there are few reports on the treatment of meningeal carcinoma in China, and no effective treatments exist. Therefore, finding new drugs to treat meningeal carcinoma is crucial in this field. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides the application of GW9662 in the treatment of meningeal carcinoma.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of GW9662 in the preparation of a medicament for treating meningeal carcinoma.

[0007] Furthermore, the meningeal carcinoma is derived from lung cancer.

[0008] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0009] Furthermore, the pharmaceutically acceptable excipients include excipients, buffers, surfactants, and preservatives.

[0010] Furthermore, the dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0011] Furthermore, the gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0012] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0013] A second aspect of the present invention provides a medicament for treating meningeal carcinoma, the medicament comprising GW9662.

[0014] Furthermore, the meningeal carcinoma is derived from lung cancer.

[0015] Furthermore, the drug also includes other active ingredients.

[0016] Furthermore, the other active ingredients include any one or more of the following: taxane, platinum-based agents, anthracycline, anthraquinone, alkylating agents, vinorelbine, nucleoside analogs, ixaprilone, eribulin, cytarabine, capecitabine, lapatinib, 5-FU, vincristine, etoposide, and methotrexate.

[0017] A third aspect of the present invention provides a method for inhibiting meningeal carcinoma cells in vitro, the method comprising administering GW9662.

[0018] Furthermore, the method described is not for therapeutic purposes.

[0019] Advantages and beneficial effects of the present invention:

[0020] This application is the first to discover that GW9662 can effectively inhibit meningeal carcinoma cells. This discovery is expected to overcome existing treatment challenges, open up new avenues for the treatment of meningeal carcinoma, significantly improve treatment efficacy, prolong patient life, and has broad application prospects in clinical practice. Attached Figure Description

[0021] Figure 1 This is a single-cell transcriptome analysis of tumor cell characteristics in cerebrospinal fluid samples from patients with meningeal carcinoma of lung adenocarcinoma (LUAD-LM) and lung tissue samples from patients with primary lung adenocarcinoma (LUAD). Specifically, 1A is a UMAP plot showing epithelial cells in cerebrospinal fluid and lung tissue samples divided into 8 subtypes; 1B is a plot showing the relative proportion of epithelial cell subpopulations in each sample; 1C is a plot showing the proportion of each cluster of epithelial cell subpopulations in CSF samples from LUAD-LM patients and lung tissue samples from LUAD patients; 1D is a GSVA plot showing the relative expression of selective gene sets in tumor cells from cerebrospinal fluid and lung tissue samples; 1E is a SCENIC analysis plot showing the differential expression of transcription factors in tumor cells from cerebrospinal fluid and lung tissue samples; 1F is a heatmap showing the relative expression of cholesterol efflux receptor and cholesterol receptor genes in each subtype of tumor cells from cerebrospinal fluid and lung tissue samples; 1G is a scoring plot of cholesterol receptor gene sets in each subtype of tumor cells from cerebrospinal fluid and lung tissue samples; and 1H is a scoring plot of cholesterol efflux gene sets in each subtype of tumor cells from cerebrospinal fluid and lung tissue samples.

[0022] Figure 2The graph shows how PPARγ inhibitors can inhibit the growth of mouse lung cancer meningeal metastases (LLC-LM-LUC). 2A shows the results of scratch assays on LLC-LM-LUC and mouse lung cancer (LLC) cells, 2B shows the results of CCK8 assays on LLC-LM-LUC and LLC cells, 2C is a quantitative bar chart of scratch assay results for LLC-LM-LUC cells, and 2D is a quantitative bar chart of scratch assay results for LLC cells. Detailed Implementation

[0023] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] This invention provides the application of GW9662 in the preparation of a medicament for treating meningeal carcinoma.

[0025] In some embodiments, GW9662 further includes pharmaceutically acceptable salts, solvates, or stereoisomers of GW9662, or combinations thereof. A pharmaceutically acceptable salt is any acid addition salt or base addition salt that is safe and effective for topical application in mammals and has the desired biological activity. The main body of a pharmaceutically acceptable salt is well known in the art. Pharmaceutically acceptable salts are preferably derived from inorganic acids or organic acids and bases. Such acidic salts include, but are not limited to, the following: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanates, hexanoates, hydrochlorides, hydrobromide, hydroiodates, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, 2-naphthalenesulfonate. Salts, nicotinic acid salts, oxalates, pyrates, pectinates, persulfates, 3-phenyl-propionates, picrates, pentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, hydrohalides (e.g., hydrochlorides and hydrobromates), sulfates, phosphates, nitrates, aminosulfonates, malonates, salicylates, methylene-bis-β-hydroxynaphthyl carboxylate, gentianates, hydroxyethylsulfonates, di-p-toluyl tartrate, ethanesulfonates, cyclohexylaminosulfonates, quinates, etc. Pharmaceutically acceptable base addition salts include, but are not limited to, those derived from alkali metal or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts containing organic bases (such as dicyclohexylamine salts, N-methyl-D-glucosamine), and salts containing amino acids such as arginine and lysine.

[0026] In some implementations, the primary origin of meningeal carcinoma is a solid tumor (e.g., brain tumors such as glioblastoma or medulloblastoma), breast tumors (e.g., breast tumors identified as HER2-positive, breast tumors identified as HER2-negative, breast tumors identified as ER-positive, breast tumors identified as ER-negative, breast tumors identified as PR-positive, breast tumors identified as PR-negative, or breast tumors identified as triple-negative), lung tumors such as lung cancer, gastrointestinal tumors, or malignant melanoma.

[0027] In some implementations, the primary origin of meningeal carcinoma is a liquid tumor (e.g., leukemia or lymphoma).

[0028] In some implementations, the primary origin of meningeal carcinoma is unknown (also referred to as cancer of unknown primary origin or occult cancer).

[0029] The drug also includes pharmaceutically acceptable excipients.

[0030] In some embodiments, pharmaceutically acceptable excipients include all solvents, diluents, buffers (e.g., neutral buffered saline, or optionally Tris-HCl, acetate, or phosphate buffers), solubilizers (e.g., polysorbate 80), colloids, dispersion media, solvents, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, humectants, stabilizers, emulsifiers, sweeteners, colorants, flavoring agents, aromatizers, thickeners, substances that provide storage effects, coating agents, antifungal agents, and preservatives (e.g., Thimerosal). TM Medium and reagents used in the formulation of pharmaceuticals include benzyl alcohol, antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, and extenders (e.g., lactose, mannitol). The use of such mediums and reagents in pharmaceutical formulations is well known in the art. Their use in pharmaceuticals may be considered unless any conventional medium or reagent is incompatible with the active ingredient.

[0031] The dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0032] In some embodiments, the gastrointestinal dosage form includes tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release formulations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0033] In some embodiments, the non-gastrointestinal dosage form includes injectable dosage form, respiratory dosage form, cavity dosage form, mucosal dosage form, and skin dosage form.

[0034] In some embodiments, the injectable dosage forms include, but are not limited to, various injectables such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and patches; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0035] The drug also includes other active ingredients.

[0036] In some embodiments, other active ingredients include, but are not limited to: abaricicliz, aldehyde interleukin, alenmab, retinoic acid, allopurinol, hexamethylmelamine, amifostine, anaproxine, anatozolazole, arsenic trioxide, asparaginase, azacitidine, and live BCG bacteria. Live), bevacizumab, bexarotin, bleomycin, bortezomib, bortezomib, busulfan, busulfan, calotestosterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, cloracine, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, radiobiotic D, dalteparin (e.g., sodium), afadabepoetin, dasatinib, doxorubicin, daunorubicin, decitabine, dentine interleukin, dentine interleukin-toxin conjugate, dextromethorphan, docetaxel, doxorubicin, drotaldone propionate, eculizumab, epirubicin (e.g., HCl), epoetin, erlotinib, estradiol, etoposide (e.g., phosphate), exemestane, fentanyl (e.g., citrate), filgrastim, fluorouracil, fludarabine, fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine (e.g., HCl), gimmick, ozomecine, goserelin (e.g., acetate), histamine relin (e.g., acetate), hydroxyurea, teimomab, idarubicin, ifosfamide, imatinib (e.g., mesylate), interferon alpha-2b, irinotecan, lapatinib xylenesulfonate, lenalidomide, letrozole, leucovorin, leuprolide Lin (e.g., acetate), levamisole, lomustine, CCNU, dichloromethyldiethylamine (nitrogen mustard), medroxyprogesterone acetate, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nannologone phenylpropionate, nerabine, romosumab, olprene, interleukin, oxaliplatin, paclitaxel, parivmin, pamidronate, panitumumab, pegaspargase, pegaspargase, ethylene glycol-modified filgrastim, pegylated interferon alpha-2b, pemetrexed (e.g., disodium), pentostatin, piperobromane, procamycin (photomycin), porphyrin (e.g., Sodium), methylbenzylhydrazine, quinacrine, raburicase, rituximab, samostin, sorafenib, streptozotocin, sunitinib (e.g., maleate), talc, tamoxifen, temozolomide, teniposide (VM-26), testinolide, thalidomide, thioguanine (6-TG), thiotepa, thiotepa, thiotepa, topotecan (e.g., HCl), toremifene, tosimomab / I-131 (tosimomab), trastuzumab, trastuzumab, retinoic acid (ATRA), uracil mustard, penoxuridine, vincristine, vinorelbine, vorinostat, zoledronic acid, and zoledronic acid.

[0037] In some embodiments, the dosage of the drug described in this application depends on several factors, including: the method of administration, the severity of the disease, the treatment or prevention of cancer, and the age, weight, and health status of the subject to be treated.

[0038] In some embodiments, the administration of the drug is not limited to a specific pattern of administration, dosage, or frequency. The drug may be given to the subject in a single dose or in multiple doses. For example, the drug may be administered once a week, or once every 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is understood that for any given subject, the specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the drug. For example, if a lower dose does not provide sufficient activity in the treatment of the disease or condition described in this application (e.g., meningocarcinomatosis), the dose of the drug may be increased. Conversely, if the disease (e.g., meningocarcinomatosis) is reduced or eliminated, the dose of the drug may be decreased.

[0039] While the attending physician will ultimately determine the appropriate dosage and regimen, the therapeutically effective dose of the drug described in this application may be in the range of, for example, 0.0035 μg to 20 μg / kg body weight / day or 0.010 μg to 140 μg / kg body weight / week. Desirably, the therapeutically effective dose is in the range of 0.025 μg to 10 μg / kg administered daily, every other day, or twice weekly, for example, at least 0.025, 0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 μg / kg body weight. In addition, the effective therapeutic dose can be administered weekly, every other week, every three weeks or monthly, from 0.05 μg to 20 μg / kg, for example, in the range of at least 0.05, 0.7, 0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0 or 18.0 μg / kg body weight.

[0040] In some embodiments, an effective amount refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to such a disease, disorder, and / or condition according to a therapeutic dosage regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of a disease, disorder, and / or condition, and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will recognize that an effective amount does not actually require successful treatment in a particular individual. Rather, an effective amount can be an amount that, when administered to a patient requiring such treatment, provides a particularly desired pharmacological response in a substantial number of subjects. Specifically, it is understood that a particular subject may be difficult to treat with respect to an effective amount. To cite just one example, a difficult-to-treat subject may have low bioavailability to the point that clinical efficacy is unattainable. In some embodiments, reference to an effective amount may refer to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will recognize that, in some embodiments, the effective amount can be formulated and / or administered in a single dose. In some embodiments, the effective amount can be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0041] In some embodiments, any of these strategies can be pursued to achieve controlled release, wherein the release rate exceeds the metabolic rate of the drug under consideration. In some embodiments, controlled release is achieved through a variety of dosage form parameters and components, including, for example, appropriate selection of various types of controlled-release drugs and coatings. Examples include single or multi-unit tablets or capsules, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.

[0042] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0043] Example

[0044] I. Experimental Methods

[0045] 1. Single-cell sequencing data analysis

[0046] 1.1 Collect patient information and samples

[0047] Four patients diagnosed with LUAD-LM and without other cancers were included in the study. Cerebrospinal fluid samples were collected from patients before treatment for 10×Genomics scRNA-seq analysis. All human samples used in this study were collected from the Second Hospital of Hebei Medical University. Informed consent and the proposed study were approved by the Institutional Review Committee of the Second Hospital of Hebei Medical University. The lung tissue samples from which the 10×Genomics scRNA-seq data of the four patients with lung adenocarcinoma (carcinoma in situ) were obtained were from the GEO (https: / / www.ncbi.nlm.nih.gov / geo / ) database (GSE123904).

[0048] 1.2 Preparation of single-cell suspension

[0049] After ex vivo, tissues were preserved in GEXSCOPE tissue preservation solution (Singleron) and transported to the Synergen laboratory within 72 hours using ice packs. Samples were washed three times with Hanks balanced salt solution (HBSS, Gibco, Cat. No. 14025-076) and cut into 1–2 mm fragments. The tissue fragments were then continuously agitated with 2 ml of GEXSCOPE tissue separation solution (Singleron) in 15 ml centrifuge tubes (Falcon, Cat. No. 352095). Cells were passed through a 40 μm sterile filter (Falcon, Cat. No. 352340) and centrifuged at 300 g (Eppendorf, 5810R) for 5 minutes. The supernatant was then removed, and the pellet was resuspended in 1 ml of PBS (Hyclone, catalog number SA30256.01). To remove red blood cells, which are typically an important component of the generated cells, 2 mL of RBC lysis buffer (Roche, catalog number 11 814 389 001) was added to the cell suspension according to the manufacturer's protocol. The cells were centrifuged at 500×g for 5 minutes at 4°C and then resuspended in PBS (Hyclone, catalog number SA30256.01). Samples from the cell mixture were stained with trypan blue (Bio-RAD, Cat. No. #1450013) and counted under a microscope (Nikon, ECLIPSE Ts2) to achieve a concentration of 1×10⁻⁶. 5 1 cell / mL, and once the cell viability exceeds 80%, subsequent sample processing can proceed.

[0050] The patient gave informed consent for cerebrospinal fluid (CSF) collection. The sample was separated into cell-free CSF and CSF cellular contents: the entire CSF sample was centrifuged at 600g (Eppendorf, 5810R) for 5 minutes. Cells were precipitated at 4°C, and the supernatant was preserved as cell-free CSF. The cell pellet was resuspended and washed twice with PBS (Gibco, USA) supplemented with 0.4% bovine serum (Gibco, USA), and cell counting was performed immediately to achieve a cell concentration of approximately 1 × 10⁻⁶ cells / mL. 5 Cells / ml

[0051] 1.3 Single-cell RNA-seq sequencing

[0052] Prepared in PBS at a concentration of approximately 1 × 10⁻⁶ 5 Single-cell suspensions were prepared at 1 cell / ml. The single-cell suspensions were then loaded onto a microfluidic device, and scRNA-seq libraries were constructed using the GEXSCOPE Single-Cell RNA Library Kit (Singleron Biotechnologies) according to the Singleron GEXSCOPE protocol. This included cell lysis, mRNA capture, cell tagging (barcoding), and mRNA (UMI). The mRNA was reverse transcribed into cDNA and amplified, and finally, the cDNA was fragmented. Single libraries were diluted to 4 nM and pooled for sequencing. Sequencing was performed on an Illumina HiSeq X using 150 bp paired-end reads. Raw reads were processed using fastQC and fastp to remove low-quality reads. Poly-A tails and adapter sequences were removed using cutadapt. After quality control, reads were mapped to the reference genome GRCh38 using STAR. Gene expression levels and UMI counts were statistically analyzed using the FeatureCounts function. Expression matrix files were generated based on gene expression levels and UMI counts for subsequent analysis.

[0053] 1.4 Raw Data Reading

[0054] Raw scRNA-seq reads were used to generate a gene expression matrix. In short, the raw reads were first processed using fastQC (Andrews et al., 2010) v0.11.4 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) and fastp (Chen et al., 2018) to remove low-quality reads, and cutadapt (Martin 2011) was used to trim poly-A tails and adapter sequences. After extracting cell barcodes and UMIs, the reads were mapped to the reference genome GRCh38 using STAR (Daehwan et al., 2013) v2.5.3a. The number of UMIs and gene expression levels for each cell were obtained using FeatureCounts (Liao et al., 2014) v1.6.2 software and used to generate the expression matrix file for subsequent analysis.

[0055] 1.5 Quality Control, Dimensionality Reduction, and Clustering

[0056] Prior to analysis, cells were filtered based on UMI counts below 30,000 and gene counts between 200 and 5,000, with cells containing more than 20% mitochondrial content removed. After filtering, functions in Seurat v2.3 (Satija et al., 2015) were used for dimensionality reduction and clustering. All gene expression was then normalized and scaled using the Normalize Data and Scale Data functions, and the top 2000 hypervariable genes were selected for PCA analysis using the Find Variable Features function. Using the top 20 principal components, the principal components were grouped into multiple clusters using FindClusters. Batch effects between samples were removed using the Harnomy algorithm (Korsunsky et al., 2019). Finally, the UMAP algorithm was used for visualization in two-dimensional space.

[0057] 1.6 Differentially expressed genes (DEGs) analysis

[0058] To identify differentially expressed genes (DEGs), a Wilcox-based nonparametric test was used. The SeuratFindMarkers function was employed with default parameters, selecting genes expressed in more than 10% of cells within a cluster and with a mean log(Fold Change) > 0.25 as DEGs. For cell type annotation within each cluster, the expression of classic markers identified in the DEGs was combined with literature data, and Seurat-generated heatmaps, dot plots, and violin plots were used to display the marker expression for each cell type. Double-cell pairs identifying expression markers for different cell types were manually filtered out.

[0059] 1.7 Pathway Enrichment Analysis

[0060] To investigate the potential functions of DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the "clusterProfiler" R package (Yu et al., 2012). Pathways with P < 0.05 were considered significantly enriched. The Gene Ontology gene set included molecular function (MF), biological process (BP), and cellular component (CC) categories as references. Protein-protein interactions (PPIs) of DEGs in each cluster were predicted based on known gene interactions with relevant GO terms in StringDB v1.22.0.

[0061] 1.8 Inference of Gene Regulatory Networks

[0062] To analyze transcription factor regulatory networks, SCENIC analysis was performed using scRNA expression matrices and transcription factors via Animal TFDB (Aibar S et al., 2017). Regulatory networks were predicted using the GENIE3 package based on the co-expression of regulators and targets. The RcisTarget package was used to search for transcription factor binding motifs in the data. Genes involved in predicting regulatory networks were defined as a gene set, and AUC values ​​were calculated using the AUCell package to assess the activity of regulatory networks in cells.

[0063] 1.9 Genomic Variation Analysis (GSVA)

[0064] Gene set variation analysis (GSVA) ​​was performed using the R package GSVA (1.40.1) to detect the activation status of 50 marker gene sets in the molecular signature database (https: / / www.gsea-msigdb.org / gsea / msigdb).

[0065] 1.10 Quantification and statistical analysis

[0066] Differences between two groups were analyzed using a two-tailed Student's t-test or a Wilcoxon Rank-Sum test. Pairwise comparisons among multiple groups were performed using one-way ANOVA or the Package Dunn test (v1.3.5), with the Kruskal-Wallis test applied. A p-value < 0.05 was considered statistically significant.

[0067] 2. Cell Scratch Assay

[0068] Items: six-hole board, pen, ruler, gun tip

[0069] 1) Draw 5 lines on the bottom surface of the six-hole plate (0.5-1cm apart).

[0070] 2) Divide the six-well plates into two groups (LLC cell group and LLC-LM-LUC cell group), with three wells in each group, and add 5×10⁻⁶ cells to each well. 5 Add 2 ml of culture medium to each LLC cell or LLC-LM-LUC cell, and incubate at 37°C with 5% CO2 for 24 hours until the cells adhere to the culture vessel. The cells will be fully colonized on the second day.

[0071] 3) Remove the six-well plate and use a (200μl) small pipette tip to scribble three lines perpendicular to the well plate.

[0072] 4) Rinse each well three times with sterile PBS to remove floating cells, add 2 ml of serum culture medium to each well, and incubate at 37°C and 5% CO2.

[0073] 5) Remove the six-well plate at 6h, 12h and 24h, observe cell growth and take pictures under an optical microscope.

[0074] 6) ImageJ software calculates the scratch width for each field of view.

[0075] 3 CCK8 Experiment

[0076] The inhibitory effect of GW9662 on the growth of LLC-LMC-LUC cells was analyzed using a Cell Proliferation and Virulence Assay (CCK-8) kit. The specific steps were as follows: When the LLC-LMC-LUC cell density in the culture flask reached 80%-90%, the culture medium was discarded, and the cells were washed twice with PBS. 0.5 ml of trypsin was added to digest the LLC-LMC-LUC cells for 3 min, followed by the addition of 2 ml of culture medium to terminate the digestion. The cells were collected by centrifugation at 500 r / min for 5 min, and sufficient culture medium was added to evenly disperse the cells before seeding them in 96-well plates (cell density: 1×10⁶ cells / well). 5 / well). Incubate at 37℃ for 24 h in a cell culture incubator. Aspirate the culture medium with a pipette and add culture medium containing different concentrations of GW9662 (0, 10, 20, 30, 40, 50, 60, and 70 μM) to each well, and continue incubation for another 24 h. Aspirate the culture medium, wash twice with PBS, add 10 μL of CCK-8 solution to each well, incubate for 2 h, and then measure the OD value of each well using a microplate reader (detection wavelength 450 nm) and record the results. Calculate cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100, where As = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8, and the test compound), Ab = absorbance of blank wells (absorbance of wells containing culture medium and CCK-8), and Ac = absorbance of control wells (absorbance of wells containing cells, culture medium, and CCK-8).

[0077] The inhibitory effect of GW9662 on LLC-LUC cell growth was analyzed using a Cell Proliferation and Toxicity Assay (CCK-8) kit. The specific steps were as follows: When the LLC-LUC cell density in the culture flask reached 80%-90%, the culture medium was discarded, and the cells were washed twice with PBS. 0.5 ml of trypsin was added to digest the LLC-LUC cells for 3 min, followed by the addition of 2 ml of culture medium to terminate the digestion. The cells were collected by centrifugation at 500 rpm for 5 min, and sufficient culture medium was added to evenly disperse the cells before seeding them in 96-well plates (cell density: 1×10⁶ cells / well). 5 / well). Incubate at 37℃ for 24 h in a cell culture incubator. Aspirate the culture medium with a pipette and add culture medium containing different concentrations of GW9662 (0, 10, 20, 30, 40, 50, 60, and 70 μM) to each well, and continue incubation for another 24 h. Aspirate the culture medium, wash twice with PBS, add 10 μL of CCK-8 solution to each well, incubate for 2 h, and then measure the OD value of each well using a microplate reader (detection wavelength 450 nm) and record the results. Calculate cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100, where As = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8, and the test compound), Ab = absorbance of blank wells (absorbance of wells containing culture medium and CCK-8), and Ac = absorbance of control wells (absorbance of wells containing cells, culture medium, and CCK-8).

[0078] 4. Experimental Materials and Instruments

[0079] Fetal bovine serum (FBS) biological reagent, Uruguay, South America; DMEM incomplete culture medium biological reagent, Beijing Solarbio Science & Technology Co., Ltd.; PBS biological reagent, Beijing Solarbio Science & Technology Co., Ltd.; Trypsin biological reagent, Beijing Solarbio Science & Technology Co., Ltd.; CCK8 kit biological reagent, Shijiazhuang Zhibao Biotechnology Co., Ltd.; Dimethyl sulfoxide (DMSO) ≥99%, Aladdin Co., Ltd.; LLC-LUC cells, ATCC Cell Company; LLC-LMC-LUC, donated by the Institute for Translational Brain Science, Fudan University.

[0080] instrument:

[0081] Electronic balance FA2104N, Shanghai Jingke Tianmei Instrument Co., Ltd.; Benchtop high-speed centrifuge TG16G, Changzhou Jintan Gaoke Instrument Factory; Inverted fluorescence microscope IX71 Olympus; Varioskan LUX multi-functional microplate reader, Thermo Fisher Scientific.

[0082] II. Experimental Results

[0083] Single-cell sequencing was performed on CSF specimens from four patients with meningeal carcinoma originating from lung adenocarcinoma. Compared with the four primary lung adenocarcinoma tissues, the PI3K / AKT signaling pathway was upregulated in tumor cells of the meningeal carcinoma CSF, indicating that the PI3K / AKT signaling pathway is involved in the development and progression of malignant tumor cell metastasis to the meninges; LDL receptor expression was increased, which means that tumor cells have increased cholesterol uptake; at the same time, the expression of cholesterol efflux transporters ABCA1 and ABCG1 was downregulated, resulting in reduced cholesterol efflux. Upregulation of cholesterol metabolism pathways led to increased expression of the cholesterol metabolism regulatory transcription factor PPARγ. Figure 1 It is speculated that, compared with primary lung adenocarcinoma tissue, tumor cells in the CSF of meningeal carcinoma patients tend to increase cholesterol accumulation through the PI3K / AKT signaling pathway, while simultaneously activating PPARγ to store cholesterol as lipid droplets, thereby preventing intracellular lipid overload. These results suggest that tumor cells may adapt to the CSF microenvironment by regulating cholesterol storage and utilization.

[0084] Different concentrations of GW9662 were used to treat mouse lung cancer meningeal metastases (LLC-LM-LUC) and mouse lung cancer cells (LLC-LUC). The results showed that GW9662 significantly inhibited the growth rate of LLC-LM-LUC cells, indicating that GW9662 has a therapeutic effect on meningeal carcinoma. Figure 2 ).

[0085] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of GW9662 in the preparation of drugs for treating meningeal carcinoma.

2. The application according to claim 1, characterized in that, The meningeal carcinoma is a symptom of lung cancer.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, Pharmaceutically acceptable excipients include excipients, buffers, surfactants, and preservatives.

5. The application according to claim 4, characterized in that, The dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

6. The application according to claim 5, characterized in that, The gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets. Preferably, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

7. A drug for treating meningeal carcinoma, characterized in that, The drug includes GW9662; Preferably, the meningeal carcinoma originates from lung cancer.

8. The medicament according to claim 7, characterized in that, The drug also includes other active ingredients.

9. The medicament according to claim 8, characterized in that, The other active ingredients include any one or more of the following: taxane, platinum-based agents, anthracycline, anthraquinone, alkylating agents, vinorelbine, nucleoside analogs, ixaprilone, eribulin, cytarabine, capecitabine, lapatinib, 5-FU, vincristine, etoposide, and methotrexate.

10. A method for inhibiting meningeal carcinoma cells in vitro, characterized in that, The method includes applying GW9662.

Images