Use of fty720 as a pp2a phosphatase activator for the preparation of a medicament for the treatment of neurofibromatosis type i

Abstract

The application discloses application of FTY720 as a PP2A phosphatase activator in preparation of a medicine for treating type I neurofibromatosis. The application first uses FTY720 for treatment of type I neurofibromatosis, and proves that FTY720 significantly inhibits formation of neurofibromas by non-specifically activating PP2A phosphatase. In-vivo experimental results show that FTY720 as a single drug can significantly inhibit tumor growth, and a synergistic effect is presented when FTY720 is combined with a MEK inhibitor, and tumor growth is almost completely inhibited. In-vitro cell experiments show that FTY720 as a single drug or in combination with MEKi treatment can inhibit tumor Schwann cells from forming tumor spheres, inhibit cell proliferation and migration, and induce tumor cell apoptosis. The application overcomes the drug resistance problem existing in the prior art MEK inhibitor, and provides a new treatment strategy for type I neurofibromatosis. FTY720 is an FDA-approved drug, and has good safety and drugability, and has high clinical conversion potential.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of FTY720 as a PP2A phosphatase activator in the preparation of a drug for treating neurofibromatosis type I. Background Technology

[0002] Neurofibromatosis type 1 (NF1) is an autosomal dominant inherited disease caused by mutations in the NF1 gene, with multiple neurofibromas being a key clinical feature. Currently, targeted kinase inhibitors (such as MEK inhibitors, MEKi) are a hot research topic for treating NF1-related neurofibromas. However, existing targeted kinase inhibitor therapies have significant limitations: on the one hand, some patients exhibit primary resistance to MEKi; on the other hand, acquired resistance develops as tumors regrow after discontinuation due to side effects. Therefore, there is an urgent need to develop novel treatment strategies to overcome the challenge of kinase inhibitor resistance.

[0003] Protein phosphatase 2A (PP2A) is an important serine / threonine phosphatase that negatively regulates various abnormally activated kinase signaling pathways through dephosphorylation. Previous studies have found somatic mutations in the PPP2R3A gene, encoding the B'' subunit of PP2A, in some neurofibromatosis tissues (detection rate approximately 30%). Simultaneously, the expression levels of other subunits of the PP2A complex (such as structural subunit A and catalytic subunit C) and the overall phosphatase activity are significantly lower than in normal neural tissue. These results suggest that downregulation of PP2A activity may be involved in the occurrence and development of neurofibromatosis, and restoring PP2A function may be a novel strategy for treating NF1.

[0004] FTY720 (fingolimod) is a drug approved by the U.S. Food and Drug Administration for the treatment of multiple sclerosis. Currently, there are no reports on the use of FTY720 in the treatment of neurofibromatosis type I or its application as a PP2A activator in in vivo animal models of neurofibromatosis.

[0005] In conclusion, developing a treatment regimen based on a PP2A activation strategy with FTY720 as the active ingredient, especially for the dosage and method of administration to type I neurofibromatosis, has potential application value and innovation. Summary of the Invention

[0006] The purpose of this invention is to provide the application of FTY720 as a PP2A phosphatase activator in the preparation of drugs for the treatment of neurofibromatosis type I, aiming to overcome the primary and acquired drug resistance problems of existing MEK inhibitors and provide a new and effective treatment strategy for neurofibromatosis type I.

[0007] To achieve the above-mentioned objectives, this invention provides the use of FTY720 as a PP2A phosphatase activator in the preparation of a medicament for treating neurofibromatosis type I.

[0008] Furthermore, the drug also contains a MEK inhibitor, and the FTY720, in combination with the MEK inhibitor, is used to treat neurofibromatosis type I.

[0009] Preferably, the MEK inhibitor is one or more of the clinically available MEK kinase inhibitors.

[0010] Preferably, the dosage of FTY720 is 5 mg / kg body weight, the administration method is gavage, the administration regimen is 5 consecutive days followed by 2 days of rest, and the total treatment cycle is 8 weeks.

[0011] Preferably, in the combination therapy, the dosage of FTY720 is 5 mg / kg body weight, the dosage of MEK inhibitor is 25 mg / kg body weight, both are administered by gavage, the dosing regimen is 5 consecutive days followed by 2 days of rest, and the total treatment period is 8 weeks.

[0012] Furthermore, the type I neurofibromatosis manifests as neurofibromas, and the drug exerts its effects through one or more of the following mechanisms: (1) inhibiting the proliferation of neurofibromatosis Schwann cells; (2) inhibiting the migration of neurofibromatosis Schwann cells; (3) inhibiting tumor spheroid formation; (4) inducing neurofibromatosis cell apoptosis; and (5) reducing the number of neurofibromas.

[0013] To achieve the above-mentioned objective, the present invention provides a pharmaceutical composition for treating neurofibromatosis type I, the pharmaceutical composition comprising PP2A phosphatase activator FTY720 and a pharmaceutically acceptable carrier or excipient.

[0014] Preferably, the dosage form of the pharmaceutical composition is an oral preparation, a gavage preparation, a tablet, or a capsule.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to apply the PP2A phosphatase activator FTY720 to the treatment of type I neurofibroma, and confirms that by non-specifically activating the PP2A complex involving multiple B subunits, the formation of neurofibroma can be significantly inhibited; this discovery opens up a new direction for drug targets in the treatment of NF1-related tumors, and gets rid of the single intervention mode of traditional kinase inhibitors.

[0016] (2) The present invention has confirmed through cell model experiments and in vivo animal experiments that FTY720 alone can inhibit the proliferation and migration of tumor Schwann cells, significantly reduce the formation of tumor spheres by tumor Schwann cells, and significantly reduce the number of neurofibromas in mice; thus, it shows that FTY720, as a PP2A activity restorer, has the potential to be used alone to treat type I neurofibromas.

[0017] (3) The combination therapy (FTY720 + MEKi) provided by the present invention can more significantly inhibit tumor cell proliferation, migration and tumor spheroid formation, and effectively induce tumor cell apoptosis compared with MEKi alone or FTY720 alone. This synergistic effect helps to overcome the primary and acquired drug resistance problems faced by MEK inhibitors in clinical practice and improve the overall treatment effect.

[0018] (4) In the DhhCre;Nf1 flox / flox autosomal neurofibroma mouse model, the efficacy of gavage administration for 8 weeks was systematically evaluated, and statistically valid data with n≥8 in each group were obtained, and effective evaluation indicators such as tumor reduction were clarified. The administration method and dosage regimen provide a reliable experimental basis for subsequent clinical trials.

[0019] (5) Although the present invention uses a non-specific PP2A activator, it has confirmed the inhibitory effect of the overall restoration of PP2A phosphatase activity on neurofibroma. This result lays a theoretical foundation for the future screening or design of precise activators that target specific PP2A holoenzyme complexes (such as PP2A containing specific B subunits), and promotes the personalized and precise development of NF1 treatment.

[0020] (6) FTY720 is an FDA-approved drug (for multiple sclerosis), and its pharmacokinetic characteristics, safety and human tolerance are relatively well understood. Repurposing this old drug for the treatment of neurofibromatosis type I can significantly shorten the drug development cycle and reduce the risk of clinical translation. Attached Figure Description

[0021] Figure 1 Representative microscopic images showing the inhibitory effect of FTY720 alone and in combination with MEK inhibitors on the formation of tumor spheres in human neurofibromatosis cells; Figure 2 A quantitative statistical graph showing the effect of FTY720 alone and in combination with MEK inhibitors on the number of tumor spheres formed in human neurofibromatosis cells; Figure 3Figure 1 shows the inhibitory effect of FTY720 alone and in combination with MEK inhibitors on the proliferation of Schwann cells (CCK8 assay); (A) shows the proliferation of immortalized normal Schwann cells in the drug-treated control group; (B) shows the proliferation of immortalized neurofibroma Schwann cells in the drug-treated experimental group; (C) shows the proliferation of primary Schwann cells extracted and cultured from human neurofibroma tissue treated with the drug. Figure 4 Figure 1 shows the results of Annexin V / PI fluorescence staining to detect the effect of FTY720 alone and in combination with MEK inhibitors on Schwann cell apoptosis; (A) is a representative microscopic image; (B) is a statistical analysis of the apoptosis results of immortalized normal Schwann cells after different drug treatments; (C) is a statistical analysis of the apoptosis results of immortalized neurofibroma Schwann cells after different drug treatments. Figure 5 Representative microscopic images (A) and quantitative statistical graphs of cell migration rate (B) for scratch assay to detect the effect of FTY720 alone and in combination with MEK inhibitors on the migration ability of primary Schwann cells extracted from human tumor tissue. Figure 6 This is a schematic diagram comparing the inhibitory effects of PP2A activation on tumor formation in vivo in Example 2; Figure 7 This is a statistical chart showing the reduction in the number of tumors formed in vivo by activating PP2A in Example 2. Detailed Implementation

[0022] To better understand the technical solution of the present invention, the present invention will be further described below with reference to specific embodiments. These examples are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0023] The MEK inhibitors used in the following examples are selumetinib (development code AZD6244, purchased from MedChemExpress, catalog number HY50706), abbreviated as MEKi in the accompanying drawings; and FTY720 (fingolimod, purchased from MedChemExpress, catalog number HY-12005).

[0024] Example 1: Inhibitory effects of FTY720 alone and in combination with MEK inhibitors on tumor spheroid formation and Schwann cell proliferation in human neurofibromatosis cells. 1. Establishment and identification of cell models (1) Cell source and culture conditions The immortalized normal Schwann cells used in this embodiment ( The two cell lines, 05.5 and immortalized neurofibroma Schwann cells, were purchased from the American Type Culture Collection (ATCC) and subsequently provided by Shanghai Ninth People's Hospital. Identification and characterization of these two cell lines can be found in the relevant literature (PMID: 27617404).

[0025] Schwann cell precursor cells were obtained from surgically removed tumor tissue of human patients with plexiform neurofibromas who had received ethical approval and signed informed consent forms. These precursor cells were then used to culture primary Schwann cells and tumor spherocytes. The specific steps were as follows: Fresh tumor tissue was minced to approximately 1 mm. 3 The cells were placed in L-15 dissociation medium containing collagenase I and dispersase II, and digested in a shaker at 37°C for 3-4 hours. Single-cell suspensions were obtained by pipetting with a narrow-mouth pipette.

[0026] (2) Tumor spheroid cell culture: The single cells obtained above were cultured at 1×10⁻⁶ cells per cell. 5 The cells were seeded at a density of 10 cells / mL in ultra-low adsorption culture plates and cultured in suspension using stem cell culture medium (DMEM / F12 as the basal medium, with the addition of 10% B27 additive, 20 ng / mL epidermal growth factor, 20 ng / mL basic fibroblast growth factor, 5 μg / mL heparin, etc.). The cells were incubated at 37°C and 5% CO2 for 7-14 days, with half of the medium being changed every 3 days to induce the formation of tumor spheroids.

[0027] (3) Primary Schwann cell adherent culture: The single cells obtained above were cultured at 5 × 10⁶ cells per cell line. 4 Cells were seeded at a density of 1 cell / mL in poly-L-lysine-coated culture dishes and cultured in Schwann medium (DMEM as the basal medium, supplemented with 10% fetal bovine serum, 2 μM saliva, and 10 ng / mL neuroregulatory protein 1) for adherent culture. The culture was placed in an incubator at 37°C and 5% CO2, with the medium changed every 2-3 days. When the cells reached 80%-90% confluence, they were passaged or subjected to further experiments.

[0028] 2. Drug preparation and concentration selection (1) Drug preparation: FTY720 powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mmol / L stock solution, which was then aliquoted and stored at -20°C protected from light. Selmetinib powder was dissolved in DMSO to prepare a 10 mmol / L stock solution, which was then aliquoted and stored at -20°C protected from light. Before use, the solution was diluted to the required working concentration with the appropriate cell culture medium. In all experiments, an equal volume of DMSO was added to the control group to ensure that the final DMSO concentration did not exceed 0.1%.

[0029] (2) Drug concentration: Based on the preliminary experimental results, in the drug treatment experiments of immortalized Schwann cells and primary Schwann cells, the drug concentration of FTY720 was 2 μmol / L (primary Schwann cells and tumor spheroids) and 5 μmol / L (immortalized Schwann cells); the drug concentration of MEK inhibitor selumetinib was selected from 5 μmol / L (primary Schwann cells and tumor spheroids) and 8 μmol / L (immortalized Schwann cells); when used in combination, FTY720 and selumetinib were combined at the above concentrations.

[0030] 3. Tumor spheroid formation inhibition experiment (1) Experimental groups: blank control group (equal volume of DMSO); FTY720 monotherapy group (2 μmol / L); MEK inhibitor monotherapy group (5 μmol / L); FTY720 (2 μmol / L) combined with MEK inhibitor group (5 μmol / L).

[0031] (2) Experimental procedure: Take the single-cell suspension derived from the human patient plexiform neurofibroma tissue obtained above and use 3×10 4 Tumor spheroids were seeded at a density of 1 spheroid per well in 24-well ultra-low adsorption plates, with 1 ml of stem cell culture medium added to each well. FTY720 and / or the MEK inhibitor selumetinib were added to the experimental groups, while the control group received an equal volume of DMSO. The culture plates were placed in a 37°C, 5% CO2 incubator for suspension culture. Starting 48 hours after drug addition, the number of tumor spheroids in each well was counted every 12 hours. The medium was replaced with half a volume and the appropriate concentration of drug was added every 3 days.

[0032] (3) Tumor spheroid counting and statistics: After culture, observe and photograph under an inverted microscope. The criteria for tumor spheroids are: cells aggregate to form spherical structures with a diameter ≥50μm, clear boundaries, and uniform cell morphology. Count the number of tumor spheroids with a diameter ≥50μm in each well. Set up at least 3 replicates for each drug treatment, and independently repeat the experiment at least three times.

[0033] (4) Experimental results: such as Figure 1 and Figure 2 Compared with the blank control group, the number of tumor spheres formed in the FTY720 monotherapy group was significantly reduced; the inhibitory effect of FTY720 combined with MEK inhibitors on tumor sphere formation was even more significant. The results indicate that FTY720, alone or in combination with MEK inhibitors, can effectively inhibit the formation of plexiform neurofibroma tumor spheres.

[0034] 4. Cell proliferation inhibition assay (CCK8 assay) (1) Cell seeding: Take the following cells in the logarithmic growth phase: immortalized normal Schwann cells ( Immortalized neurofibroma Schwann cells (05.5); Primary Schwann cells derived from human patient tumor tissue isolated and cultured previously. The above cells were seeded at a density of 2000 cells / well in 96-well plates treated with poly-L-lysine for 1 hour, and 100 μL of the corresponding complete culture medium was added to each well (DMEM / F12 medium containing 10% fetal bovine serum was used for immortalized cells, and Schwann cell medium was used for primary Schwann cells). The plates were incubated overnight at 37°C and 5% CO2 to allow the cells to adhere.

[0035] (2) Drug treatment: After cell adhesion, discard the original culture medium and add fresh culture medium containing the corresponding concentration of drug according to the experimental groups. Experimental groups: blank control group (equal volume of DMSO), FTY720 (2 μmol / L for primary Schwann cells, 5 μmol / L for immortalized Schwann cells), MEKi (5 μmol / L for primary Schwann cells, 8 μmol / L for immortalized Schwann cells), FTY720+MEKi (2 μmol / L + 5 μmol / L for primary Schwann cells, 5 μmol / L + 8 μmol / L for immortalized Schwann cells). Each group was set up with 3 replicates. Continue culturing for 12 hours, 24 hours, and 48 hours.

[0036] (3) CCK8 detection: After the culture was completed, 10 μL of CCK8 solution was added to each well, and the mixture was gently shaken and then placed in an incubator at 37°C and 5% CO2 for 4 hours. The absorbance value (OD value) at 450 nm was measured using a microplate reader, and the absorbance value at 650 nm was used as a reference. Each drug treatment was set up with at least 3 replicates, and the experiment was independently repeated at least three times.

[0037] (4) Experimental results are as follows Figure 3 As shown: In immortalized neurofibroma Schwann cells (05.5), FTY720 monotherapy significantly inhibited cell proliferation in a time-dependent manner; the inhibitory effect was further enhanced in the group treated with FTY720 in combination with a MEK inhibitor, which was significantly better than the monotherapy group. Figure 3 B). In primary Schwann cells derived from human patient tumor tissue, FTY720 monotherapy and in combination with MEK inhibitors also exhibited significant cell proliferation inhibition. Figure 3 C). In immortalized normal Schwann cells ( In the same treatment conditions, the cell proliferation inhibition effect was weaker than that of neurofibroma Schwann cells. Figure 3 A) indicates that FTY720 and its combination therapy have a selective inhibitory effect on the proliferation of diseased cells.

[0038] 5. Apoptosis detection assay (Annexin V / PI fluorescence staining method) (1) The experimental groups were the same as those in the cell proliferation inhibition experiment. Based on the preliminary experimental results, representative concentrations were selected for the experiment: 5 μmol / L for the FTY720 monotherapy group; 8 μmol / L for the MEK inhibitor selumetinib monotherapy group; and FTY720 (5 μmol / L) + selumetinib (8 μmol / L) for the combination therapy group.

[0039] (2) Cell treatment: Immortalized normal Schwann cells in the logarithmic growth phase were taken ( Immortalized neurofibroma Schwann cells (05.5) and primary Schwann cells derived from human patient tumor tissue isolated and cultured previously were used at a concentration of 1 × 10⁻⁶. 6 Cells were seeded at a density of 1 cell / well in 6-well plates, with 2 mL of the corresponding complete culture medium added to each well. The plates were then incubated overnight at 37°C with 5% CO2. After cell attachment, the original culture medium was discarded, and fresh culture medium containing the corresponding concentration of drug was added according to the experimental groups. The plates were then incubated for another 48 hours.

[0040] (3) Annexin V / PI fluorescence staining: After drug treatment, collect the cell culture supernatant (containing suspended cells) from each group, digest adherent cells with 0.25% trypsin without EDTA, collect the cell pellet, and combine it with the supernatant. Centrifuge at 300×g for 5 minutes, discard the supernatant, and wash the cells twice with pre-cooled phosphate buffer. Resuspend the cell pellet in 100μL of 1×Annexin V binding buffer, add 5μL of Annexin V-FITC and 5μL of propidium iodide (PI) staining solution, mix gently, and incubate at room temperature in the dark for 15 minutes. After incubation, add 400μL of 1×Annexin V binding buffer, and immediately observe and photograph using a fluorescence microscope. Figure 4 A).

[0041] (4) Apoptotic cell count: Observed and counted under a fluorescence microscope: Annexin V single positive (FITC) + / PI - These are early apoptotic cells, double-positive for Annexin V and PI (FITC). + / PI + () represents late-stage apoptotic cells and necrotic cells. At least 5 fields of view were randomly selected from each group for counting, and the apoptosis rate (%) was calculated as (number of early-stage apoptotic cells + number of late-stage apoptotic cells) / total number of cells × 100%.

[0042] (5) Experimental results: such as Figure 4 As shown in A and 4C, in immortalized neurofibroma Schwann cells, compared with the blank control group, the apoptosis rate of immortalized neurofibroma Schwann cells treated with FTY720 alone was significantly increased; the MEK inhibitor selactinib alone treatment group also significantly induced apoptosis; however, the apoptosis rate induced by the combination of FTY720 and the MEK inhibitor was significantly higher than that of any single drug group, showing a synergistic or additive effect. The results observed in primary tumor Schwann cells are consistent with those in immortalized neurofibroma Schwann cells (not shown in the figure). Figure 4 As shown in A and 4B: In immortalized normal Schwann cells ( In the study, under the same treatment conditions, the apoptosis rate of FTY720 cells was significantly lower than that of neurofibroma Schwann cells, indicating that FTY720 and its combination therapy have a selective apoptosis-inducing effect on diseased cells. These results demonstrate that FTY720, alone or in combination with MEK inhibitors, can effectively induce apoptosis in neurofibroma Schwann cells.

[0043] 6. Cell migration inhibition assay (scratch assay) (1) The experimental groups were the same as those in the cell proliferation inhibition experiment. Representative concentrations were selected for the experiment: 2 μmol / L for the FTY720 monotherapy group; 5 μmol / L for the MEK inhibitor selumetinib monotherapy group; and FTY720 (2 μmol / L) + selumetinib (5 μmol / L) for the combination therapy group.

[0044] (2) Cell seeding and scratching: Primary Schwann cells derived from human patient tumor tissue obtained in the logarithmic growth phase were seeded at a rate of 6 × 10⁻⁶ cells / year. 5 Seeds were placed at a density of cells / well in 6-well plates, with 2 mL of Schwann cell culture medium added to each well. The plates were then incubated at 37°C with 5% CO2 until the cells reached 100% confluence to form a monolayer. Using a 200 μL sterile pipette tip, a vertical line was drawn across the cell monolayer in each well, ensuring the line width remained consistent. The cells were gently washed 2-3 times with phosphate-buffered saline (PFS) to remove any detached cells. Each well was then supplemented with low-serum (containing 0.5% fetal bovine serum) Schwann cell culture medium containing the appropriate drug concentration.

[0045] (3) Observation and statistics of scratch healing Immediately after scratching, photographs were taken at a fixed location under an inverted microscope to record the initial scratch width (recorded as 0 hours). The culture plates were then placed in an incubator at 37°C and 5% CO2 for further incubation. The healing status of the scratch at the same location was observed and photographed at 6, 12, and 24 hours. The scratch area or width at each time point was measured using ImageJ software. Cell migration rate was calculated using the following formula: Migration rate (%) = (Scratch area at 0 hours - Scratch area at a certain time point) / Scratch area at 0 hours × 100% (4) Experimental results Experimental results are as follows Figure 5 As shown in Figures A and 5B: In the blank control group, cells gradually migrated to the scratch area within 48 hours, the scratch gradually healed, and the migration rate significantly increased. Compared with the blank control group, the migration ability of cells in the FTY720 monotherapy group was significantly inhibited, as evidenced by a slower scratch healing rate. The MEK inhibitor selactinib monotherapy group also significantly inhibited cell migration. Notably, the inhibitory effect of FTY720 combined with the MEK inhibitor on cell migration was significantly stronger than that of any single-drug group. These results indicate that FTY720, alone or in combination with a MEK inhibitor, can effectively inhibit the migration ability of neurofibromatosis Schwann cells, and the combination therapy has a synergistic effect.

[0046] All experimental data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered statistically significant.

[0047] Example 2: In vivo efficacy evaluation of FTY720 alone and in combination with MEK inhibitors for the treatment of type I neurofibromatosis 1. Laboratory animals, drug preparation and group administration This embodiment uses the DhhCre;Nf1 flox / flox autosomal neurofibroma mouse model.

[0048] Nf1 conditional gene knockout mice ( Both the DhhCre transgenic mouse and the DhhCre transgenic mouse (Dhh-Cre) were purchased from Cyagen Biosciences (Suzhou, China). The mouse strains were designated (C57BL / 6J, CKOAIP240111HZ8) and (FVB / N, C 001335). All experimental mice were housed in an SPF-grade animal facility at 22±2°C with a 12-hour light-dark cycle and free access to food and water. All of the aforementioned mouse strains were publicly available from Cyagen (Suzhou) Biotechnology Co., Ltd. prior to this application date. DhhCre;Nf1 flox / flox genotype mice were bred through mating and, when reached 4 months of age, were randomly divided into the following 4 groups, with at least 8 mice in each group: Drug preparation: For single-drug preparation, the dosage of FTY720 (5 mg / kg) or MEKi (25 mg / kg) was calculated based on the mouse body weight and dissolved in DMSO. Solubilizers PEG-300 and Tween-80 were added, and physiological saline was added to make up the total dose (200 μL per gavage). The final composition was 10% DMSO (containing the drug solute), 40% PEG-300, and 5% Tween-80. For combined administration, the dosages of FTY720 (5 mg / kg) and MEKi (25 mg / kg) were calculated based on the mouse body weight and dissolved simultaneously in DMSO. Solubilizers and physiological saline were added according to the above proportions.

[0049] (1) Drug carrier control group (Ctrl): An equal volume of drug solvent was administered, with the solvent composition being 10% DMSO + 40% PEG300 + 5% Tween 80 + physiological saline; (2) FTY720 monotherapy group: FTY720 was administered at a dose of 5 mg / kg; (3) MEK inhibitor monotherapy group: MEK inhibitor selumetinib was administered at a dose of 25 mg / kg; (4) Combination therapy group: FTY720 (5 mg / kg) and MEK inhibitor selmetinib (25 mg / kg) were administered in combination.

[0050] All medications were administered via gavage, with each mouse receiving 0.2 mL of medication. The administration regimen consisted of 5 consecutive days of gavage followed by 2 days of rest, for a total of 8 weeks of continuous drug treatment.

[0051] 2. Animal perfusion and fixation After drug treatment, the mice were anesthetized with isoflurane until they lost sensation of pain and showed no response to toe clamping. The mice were then fixed in a supine position on a foam board, their limbs secured with tape, and the skin of the chest and abdomen disinfected with alcohol. The heart was exposed by opening the chest, and the apex was gently lifted with forceps. A small incision was made at the right atrial appendage (right atrium) to drain blood. A perfusion needle (connected to a perfusion tube) was then inserted into the aorta through the apex of the left ventricle and slowly advanced into the ascending aorta. Physiological saline was first perfused at a low flow rate of 5-10 mL / min, approximately 20-30 mL per mouse, until the liver turned white and the outflow from the right atrium was bloodless. Then, the flow was switched to 4% paraformaldehyde (PFA) for perfusion fixation, approximately 15-20 mL per mouse. After perfusion, the limbs, internal organs, and tail of the mice were removed, and residual blood was washed away with PBS. The mice were then placed whole into 50 mL centrifuge tubes, fixed in formalin solution, and stored for 24 hours or more.

[0052] 3. Decalcification treatment JYBL-1 decalcification solution was added to centrifuge tubes containing fixed mice, ensuring the mice were completely immersed in the solution. Since whole-mouse decalcification was performed, the decalcification time was set to 36 hours or more to avoid incomplete decalcification. After decalcification, the decalcification solution was discarded, and the mice were washed three times with PBS to remove any residual solution. Finally, the mice were stored immersed in PBS for subsequent tumor sampling.

[0053] 4. Tumor sampling and observation After decalcification, mice were fixed in a prone position, and the skin on their backs was disinfected with alcohol. The skin was longitudinally incised along the midline of the spine, and the muscles were dissected laterally to expose the spine. Using fine scissors and forceps, the lamina was removed layer by layer to fully expose the spinal cord and dorsal root ganglia (DRGs). Under a stereomicroscope, the connective tissue surrounding the tumor was gently dissected with fine forceps and scissors to completely remove the tumor. The removed tumor tissue was immersed in 75% ethanol, photographed, and its size, location, and morphological characteristics were observed and recorded.

[0054] 5. Experimental Results Statistical analysis was performed on tumor number, tumor volume, and other indicators in each group of mice. All statistical analyses were performed using Graphpad Prism software, and experimental data are expressed as mean ± standard error (Mean ± SEM). Results showed that compared with the drug carrier control group, administration of FTY720 alone (5 mg / kg) or the MEK inhibitor selumetinib alone (25 mg / kg) significantly inhibited the growth of neurofibromas; compared with either single-drug treatment group, the combination therapy group of FTY720 and the MEK inhibitor selumetinib (5 mg / kg + 25 mg / kg) showed a more significant inhibitory effect on tumor growth, with tumor growth almost completely inhibited (e.g., ...). Figure 6 and Figure 7 (As shown).

[0055] The results of this embodiment demonstrate that non-specific activation of PP2A phosphatase by the activator FTY720 can partially inhibit the formation of type I neurofibromatosis. Furthermore, the combined use of FTY720 and a MEK inhibitor produces a synergistic anti-tumor effect, further significantly inhibiting tumor formation. These results prove that FTY720, as a PP2A phosphatase activator, can be used alone or in combination with the MEK inhibitor sumetinib to prepare drugs for the treatment of type I neurofibromatosis, providing experimental evidence for clinical translation.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of FTY720 as a PP2A phosphatase activator in the preparation of drugs for the treatment of neurofibromatosis type I.

2. The application according to claim 1, characterized in that, The drug also contains a MEK inhibitor, and the FTY720, in combination with the MEK inhibitor, is used to treat neurofibromatosis type I.

3. The application according to claim 2, characterized in that, The MEK inhibitor is one or more of the clinically used MEK kinase inhibitors.

4. The application according to claim 1, characterized in that, The dosage of FTY720 is 5 mg / kg body weight, administered by gavage, with a dosing regimen of 5 consecutive days followed by 2 days of rest, for a total treatment period of 8 weeks.

5. The application according to claim 2, characterized in that, In the combination therapy, the dosage of FTY720 was 5 mg / kg body weight, and the dosage of MEK inhibitor was 25 mg / kg body weight. Both were administered by gavage. The dosing regimen was 5 consecutive days followed by 2 days of rest, with a total treatment period of 8 weeks.

6. The application according to claim 1 or 2, characterized in that, The drug works through one or more of the following mechanisms: (1) inhibiting the proliferation of neurofibroma Schwann cells; (2) inhibiting the migration of neurofibroma Schwann cells; (3) inhibiting tumor spheroid formation; (4) inducing neurofibroma cell apoptosis; and (5) reducing the number of neurofibromas.

7. A pharmaceutical composition for treating neurofibromatosis type I, characterized in that, The pharmaceutical composition comprises PP2A phosphatase activator FTY720 and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is an oral preparation, a gavage preparation, a tablet, or a capsule.

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