Application of quinacrine and linagliptin in treatment of glioma

Abstract

According to the application of the quinacrine and the linagliptin in treatment of glioma, the quinacrine can change the metabolic state of tumor cells, so that the sensitivity of the tumor cells to the linagliptin is increased, the treatment effect is enhanced, and the synergistic effect cannot be achieved by a single medicine. The treatment scheme of the quinacrine combined with the linagliptin is expected to become a new and effective treatment choice. The traditional Chinese medicine composition not only can improve the survival rate of a patient, but also can improve the life quality of the patient, and has important clinical application value and wide market prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an application of quinacrine combined with linagliptin in treating glioma. Background Art

[0002] Glioma, the most common primary malignant tumor of the central nervous system, accounts for approximately 30% of adult brain tumors. Its aggressive growth pattern and high recurrence rate result in a generally poor prognosis. The current standard of care utilizes maximal surgical resection combined with postoperative temozolomide-based chemoradiotherapy. For high-grade gliomas, bevacizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody, can be added to inhibit pathological vascular proliferation. For relapsed / refractory cases, second-line treatment strategies primarily involve molecular targeted intervention with the alkylating agent lomustine (CCNU) or BRAF / MEK signaling pathway inhibitors. Recent molecular classification studies have revealed that patients with isocitrate dehydrogenase (IDH) wild-type genes have significantly lower response rates to conventional chemoradiotherapy than those with IDH mutations. Molecular events such as epidermal growth factor receptor (EGFR) gene amplification and TERT promoter mutations can exacerbate treatment resistance. It is worth noting that the high heterogeneity of tumors and their interaction with epigenetic regulatory networks lead to dynamic evolution of key signaling pathways, which accelerates the process of acquired resistance to temozolomide (TMZ) chemotherapy, resulting in a 5-year survival rate of less than 10% for patients, making it difficult for glioma patients to break through the survival bottleneck.

[0003] Metabolism is a key characteristic of tumors. Tumor cells undergo metabolic reprogramming to meet their needs for rapid proliferation, invasion, and drug resistance. Tumor cells exhibit metabolic patterns that are significantly different from normal cells, including enhanced glycolysis (Warburg effect), glutamine metabolism, fatty acid synthesis, and nucleotide synthesis. In addition, changes in phospholipid metabolism are also an important component of tumor metabolism. Tumor cells reshape the structure and function of the cell membrane by enhancing the synthesis and remodeling of phospholipids to adapt to the needs of rapid cell division and proliferation. Changes in cell membrane phospholipids have a functional regulatory effect on gliomas. Cytosolic phospholipase A2 (cPLA2) breaks down phospholipids to produce fatty acids, which enter the tricarboxylic acid cycle through β-oxidation and participate in energy metabolism. Studies have found that the mesenchymal subtype marker polymerase-1 and transcript release factor (PTRF / Cavin1) stabilizes cPLA2 protein, increasing unsaturated lysophosphatidylcholine (LPC) levels. This LPC-mediated enhancement of membrane fluidity promotes tumor proliferation and suppresses immune responses. The cPLA2 inhibitor AACOCF3 blocks this process and significantly prolongs the survival of tumor-bearing mice. However, long-term cPLA2 inhibition leads to an imbalance between saturated phosphatidylcholine (PC) and LPC in glioma cells, further activating the cell membrane oncogenic receptor EGFR and transmitting oncogenic signals to drive tumor growth.

[0004] Dipeptidyl peptidase 4 (DPP4) plays a central regulatory role in the PTRF-cPLA2 lipid remodeling pathway by occupying key amino acid residues involved in c-Cbl binding to EGFR, stabilizing the EGFR protein from degradation by the ubiquitin-proteasome pathway. EGFR exons 25-28 (Ex25-28) and DPP4 amino acids 506-766 (AA506-766) are key domains of interaction. The DPP4 inhibitor linagliptin disrupts the DPP4-EGFR complex and promotes EGFR degradation, exhibiting significant synergistic effects with AACOCF3 in the treatment of GBM. Quinacrine, a member of the aminoacridine class of compounds, inhibits viral replication by intercalating between base pairs in DNA and RNA, thereby exerting its antiviral effects. Furthermore, quinacrine can increase the pH within acidic organelles, inhibiting autophagy, and acts as a potent cPLA2 inhibitor. Pharmacokinetic properties show that quinacrine can be rapidly absorbed after oral administration, has good tolerability and safety in vivo, and its biosafety is better than that of AACOCF3. It has great potential for clinical translation in combination with linagliptin for the treatment of glioma.

[0005] Mitochondria and lysosomes are two important intracellular organelles that interact through multiple mechanisms and are crucial for maintaining cellular homeostasis. High-resolution microscopy studies have revealed that the dynamic formation of inter-organelle membrane contact sites (MLCS) between mitochondria and lysosomes allows them to interact directly through a pathway distinct from mitophagy or the lysosomal degradation of mitochondria-derived vesicles. Rab7 is a small GTPase that plays a central role in mitophagy. Mitochondria-lysosome contact site tethering is also mechanistically regulated by mitochondrial proteins that promote Rab7 GTP hydrolysis, allowing bidirectional communication between mitochondria and lysosomes and regulating the dynamics of their organelle network, including mitochondrial fission. This dynamic contact and dissociation process is crucial for maintaining cellular homeostasis and provides a potential target for cancer therapy, which is characterized by highly active mitochondrial function to meet metabolic demands. Summary of the Invention

[0006] In view of this, the present invention aims to overcome the defects in the prior art and proposes an application of quinacrine combined with linagliptin in the treatment of glioma.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] The first object of the present invention is to provide a use of quinacrine combined with linagliptin in the preparation of a drug for treating gliomas, wherein gliomas include all low-grade and high-grade gliomas classified by the WHO.

[0009] The second object of the present invention is to provide a pharmaceutical composition for treating glioma, comprising: a preparation containing quinacrine and a preparation containing linagliptin.

[0010] In some specific embodiments, the mass ratio of quinacrine to linagliptin in the pharmaceutical composition is 2:1. Further preferably, the daily dosage of quinacrine is 10 mg / kg body weight, and the daily dosage of linagliptin is 5 mg / kg body weight.

[0011] In some specific embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0012] In some specific embodiments, the excipients include but are not limited to diluents, fillers, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, fillers, flavorings, preservatives, dispersants, film formers, plasticizers, pore formers, pH adjusters, opacifiers, retardants, and solvents.

[0013] In some specific embodiments, the dosage forms of the preparation containing quinacrine and the preparation containing linagliptin include but are not limited to capsules, tablets, powders, injections, oral preparations, etc.

[0014] The third object of the present invention is to provide a use of a drug combining quinacrine and linagliptin in the preparation of a drug for treating glioma.

[0015] In some specific embodiments, the drug promotes mitochondria-lysosome contact, thereby promoting the degradation of mitochondria-related proteins and inhibiting the energy metabolism process of tumor cells.

[0016] In some specific embodiments, the dosage form of the drug is a conventional dosage form in the field of pharmacy, wherein different types of drugs are packaged separately or together, and different types of drugs are in the same dosage form or different dosage forms.

[0017] The fourth object of the present invention is to provide the use of quinacrine in the preparation of a sensitizer for treating glioma with linagliptin.

[0018] In some specific embodiments, the sensitizer increases the sensitivity of tumor cells to linagliptin by changing the metabolic state of tumor cells, thereby enhancing the therapeutic effect.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) Glioma cells are prone to developing resistance to single drugs, but the combined use of quinacrine and linagliptin in the present invention is expected to overcome this problem. Quinacrine can alter the metabolic state of tumor cells, increasing their sensitivity to linagliptin and thus enhancing the therapeutic effect. This synergistic effect is not achievable with a single drug, demonstrating its creativity in solving practical clinical problems.

[0021] (2) Quinacrine and linagliptin are already marketed for the treatment of other diseases, and their pharmacological and toxicological properties are well understood, which facilitates their application in the treatment of gliomas. By recombining these two known drugs and applying them to new therapeutic areas, not only does it reduce the initial cost and time of drug development, but it also provides new ideas and strategies for glioma treatment, which is an innovative attempt in the field of drug repurposing.

[0022] (3) Given the extremely limited survival of glioblastoma patients and the efficacy bottlenecks of existing treatments, the proposed treatment regimen of quinacrine combined with linagliptin is expected to become a new and effective treatment option. It can not only improve patients' survival rate but also their quality of life, and has important clinical application value and broad market prospects.

[0023] (4) Since both quinacrine and linagliptin are already on the market, their production processes and quality control are quite mature, which will greatly facilitate the rapid translation and clinical application of this combination therapy. Moreover, quinacrine and linagliptin have accumulated rich clinical experience in their respective therapeutic fields, and their safety and tolerability are relatively good. When used in combination, by rationally adjusting the drug dosage and administration regimen, it is expected that their safety will be further improved and the occurrence of adverse reactions will be reduced, so that this innovative therapy can better benefit glioma patients and bring them new hope. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of quinacrine promoting the autophagic degradation of cPLA2;

[0025] Figure 2 This is the result of the synergistic effect analysis between quinacrine and linagliptin;

[0026] Figure 3 To compare the effects of quinacrine combined with linagliptin and AACOCF3 combined with linagliptin on mitochondria and energy metabolism;

[0027] Figure 4 These are the results of an in vivo animal study on quinacrine combined with linagliptin;

[0028] Figure 5This is the result of quinacrine combined with linagliptin promoting mitochondria-lysosome contact;

[0029] Figure 6 This is the result of quinacrine combined with linagliptin promoting mitochondrial protein degradation and inducing apoptosis. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0031] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0032] Where values are described herein as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values falling within that range, regardless of whether a specific value or sub-range is explicitly stated.

[0033] In this document, "a plurality of" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0034] In this document, the terms “preferably” and “more preferably” are only used to describe implementation methods or examples with better effects. It should be understood that they do not limit the scope of protection of the present invention.

[0035] In this document, the word "further" or the like is used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0036] In this article, the term "and / or" is used to describe an association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0037] As used herein, the term "about" means + / - 10%, preferably + / - 5%, more preferably + / - 1% of the specified value.

[0038] In this document, the terms “include,” “including,” “have,” “contain,” etc. are open-ended terms, meaning including but not limited to.

[0039] The term "combination" or "combination use" or "co-administration" or "combination drug" as used herein refers to the use of therapeutic agents included in the same or separate pharmaceutical formulations at the same time or different times.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0041] The materials and methods involved in the present invention are as follows:

[0042] (1) Cell proliferation inhibition experiment:

[0043] First, 100 μL of cell suspension was seeded into each well of a 96-well plate, with a cell population of 5,000 per well. The cells were cultured in an incubator for 24 hours to allow attachment. After cell attachment, different concentrations of the test drug were added to the wells of the plate. The plate was incubated in the incubator for a further 48 hours, followed by the addition of 10 μL of CCK-8 solution to each well. After the plate was incubated for an additional 2 hours, the absorbance of each well at 450 nm was measured using a microplate reader. The wells required for this experiment were as follows: experimental wells (As: culture medium with cells, CCK-8, test substance); control wells (Ac: culture medium with cells, CCK-8, no test substance); and blank wells (Ab: culture medium without test substance and cells, CCK-8). Cell survival rate was calculated as [(As - Ab) / (Ac - Ab)] × 100%; cell inhibition rate was calculated as [(Ac - As) / (Ac - Ab)] × 100%. Synergy scores were calculated using the Bliss model.

[0044] (2) Immunofluorescence (IF) experiment:

[0045] Drug- and DMSO-treated cells were washed twice with PBS, fixed with 4% PFA for 20 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, blocked with 10% BSA for 1 hour, and incubated with the corresponding primary antibodies overnight at 4°C. The next day, cells were stained with the corresponding secondary antibodies and nuclei were counterstained with DAPI. Slides were imaged using a laser confocal microscope.

[0046] (3) Fluorescence resonance energy transfer (FRET) experiment:

[0047] TOM20-Venus and LAMP1-mTurquoise2 were transfected into TBD-0220 cells. Drug- and DMSO-treated cells were washed twice with PBS, fixed with 4% PFA for 20 minutes, and washed three times with PBS. The cells were then mounted with DAPI-containing mounting medium, and lysosomal lifetime was measured using Leica fluorescence lifetime imaging microscopy (FLIM).

[0048] (4) Super-resolution microscopy imaging: An appropriate amount of TBD was plated onto a confocal dish and treated with drugs 24 hours later. Forty-eight hours after drug treatment, lysosomes and mitochondria were labeled in living cells using LysoBrite Lysosomal Blue fluorescent probe dye and PK MITO Deep Red dye. Finally, imaging was performed using the High Intelligent and SensitiveSIM (HIS-SIM) intelligent ultra-sensitive super-resolution microscope from Super Vision Technologies.

[0049] (5) Western blot (WB) experiment:

[0050] After drug stimulation or pretreatment, cells were washed with pre-chilled PBS and lysed with RIPA to extract protein. Protein concentration was measured using a BCA kit, and loading buffer was added to the protein and boiled. 30-50 μg of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. Blocked with skim milk, the membrane was incubated with the primary antibody overnight at 4°C. After incubation with a horseradish peroxidase-conjugated secondary antibody, the membrane was exposed to a luminescent solution. Protein absorbance was analyzed using the Chemi-docXRS+ (Bio-Red) image analysis system.

[0051] (6) Co-immunoprecipitation (Co-IP) experiment:

[0052] Collect the cells, add NP40 and lyse on ice for 30 minutes, centrifuge at 12000g for 15 minutes, and keep the supernatant for later use. In the supernatant after centrifugation, keep 80μL for each group as input. Take 5μL of the supernatant after centrifugation and use the BCA method to detect the protein concentration. Pre-clearing: Add 20μL of magnetic beads to the remaining supernatant, mix on a rocker at 4°C for 30 minutes, then place on a magnetic rack to take the supernatant. Add 5μL of target protein antibody to the supernatant after pre-clearing, and add the corresponding mouse or rabbit IgG antibody to the IgG group, shake on a rocker at 15 rpm, and incubate at 4°C overnight. Pre-activate magnetic beads: Add at least 20μL of magnetic beads to each EP tube, add 100μL of NP40+PMSF, place on a magnetic rack to discard the supernatant, and add 100μL to resuspend the magnetic beads. Add 100μL of pre-activated magnetic beads to each IP system, use a rocker at 4°C, and mix for 3 hours. Place the IP systems from different groups on a magnetic rack, remove the supernatant, and then wash the beads four times with PBST + PMFS (400 μL each time, 10 minutes each) on a rocking platform at 4°C. Add 40 μL of 2.5X Loading Buffer, boil at 100°C for 10 minutes, place on a magnetic rack, and load 20 μL of sample into each well for Western blotting.

[0053] (7) Seahorse experiment:

[0054] Cells were plated on Seahorse XF24 cell culture plates. Forty-eight hours after drug treatment, extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) were measured using a Seahorse Extracellular Flux Analyzer according to the manufacturer's instructions. Data were analyzed using Seahorse Wave Controller software and normalized to cell number.

[0055] (8) Transmission electron microscope (TEM) experiment:

[0056] Cells treated with drugs for 48 hours were fixed with 2.5% glutaraldehyde and then treated with 1% osmium tetroxide solution. The cells were then dehydrated with graded ethanol and dried with hexamethyldiazane. Tissue samples were embedded in epoxy resin, sectioned at 70 nm, and stained with 2% uranyl acetate and lead citrate. Finally, slides were sputtered with gold-palladium and imaged using an H7760 microscope.

[0057] (9) Mouse orthotopic glioma model:

[0058] CT2A cells were transfected with the luciferase fluorescent virus and subsequently passaged and expanded as normal. The cells were digested, centrifuged to obtain a cell pellet, washed twice with sterile PBS, resuspended in PBS, and counted. Four-week-old female BALB / c nude mice and C57 mice were used to establish intracranial GBM models. Each mouse was injected with 3×104 / 3μL of GBM cells transfected with the fluorescent virus. Under stereotactic guidance, 3μL of cells transfected with the luciferase fluorescent virus were injected intracranially into each mouse. The injection point relative to bregma was 2.0 mm posterior, 2.0 mm lateral, and 3.0 mm ventral. The mice were carefully observed after transplantation. Tumor growth was assessed by bioluminescence imaging on day 7 after transplantation.

[0059] (10) ELISA experiment:

[0060] Rinse tumor tissue from tumor-bearing mice with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood. After weighing, mince the tissue. Add the minced tissue to the appropriate volume of PBS containing protease inhibitors (1:9 weight-to-volume ratio) in a glass homogenizer and grind thoroughly on ice. Centrifuge the homogenate at 5000g for 10 minutes, and collect the supernatant for analysis. Remove the plate from the kit, which has been equilibrated at room temperature for 10 minutes. Add 100 μL / well of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 60 minutes. Add biotinylated antibody: Remove the plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well. Cover with a film sealer and incubate at 37°C for 60 minutes. Wash the plate: Discard the liquid, add 300μL of 1x wash solution to each well, let it stand for 1 minute, shake off the wash solution, pat dry on absorbent paper, and repeat the wash three times. Add enzyme conjugate working solution: Add 100μL of enzyme conjugate working solution to each well, cover with a sealing film, and incubate at 37℃ for 30 minutes. Wash the plate: Discard the liquid and wash the plate 5 times. The washing steps are the same as above. Add substrate: Add 90μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37℃ in the dark for 15 minutes. Add stop solution: Remove the ELISA plate and directly add 50μL of stop solution to each well. Immediately measure the OD value of each well at a wavelength of 450nm. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as the correction value. Plot the standard curve of the four-parameter logistic function on double logarithmic coordinate paper with concentration as the horizontal axis and OD value as the vertical axis.

[0061] The present invention will be described in detail below with reference to the embodiments.

[0062] Example 1: Quinacrine promotes the autophagic degradation of cPLA2 by promoting the binding of p62 to cPLA2

[0063] Quinacrine (Qc) has been reported to be an inhibitor of cPLA2, but the specific mechanism is unclear. Therefore, we investigated the regulatory mechanism of quinacrine on cPLA2. Given that quinacrine is also an autophagy inhibitor, we first investigated its effect on autophagic flux. The results showed that quinacrine could upregulate the protein levels of p62 and LC3-Ⅱ in GBM cells in a temporal gradient ( Figure 1 A, Figure 1 B) The increase in LC3-II levels may be due to increased autophagosome formation or inhibition of protein degradation. To distinguish these two possibilities, we first examined key genes that play an important role in autophagosome maturation. Figure 1 As shown in C: The total protein levels of these genes remained unchanged after quinacrine treatment, thus excluding the possibility that quinacrine induces autophagy and further confirming the role of quinacrine in inhibiting autophagic flux. We observed that in the early stage of treatment (0-24h), quinacrine did not significantly affect the protein level of LC3-Ⅱ, and only after 24 hours did it significantly increase the protein level of LC3-Ⅱ, indicating that quinacrine did not affect the autophagic flux in the early stage of treatment ( Figure 1 A, Figure 1 B). We also observed that the intracellular p62 level was significantly upregulated in the early stage of quinacrine action ( Figure 1 A, Figure 1 B), which suggests that quinacrine may promote the degradation of cPLA2 through the autophagy pathway.

[0064] To verify this, we treated GBM cells with the protein synthesis inhibitor cycloheximide (CHX) or CHX combined with quinacrine in a time-dependent manner. Immunoblotting results showed that the degradation rate of cPLA2 in the combined drug group was faster than that in the single drug group ( Figure 1 D, Figure 1 E). Given that the ubiquitin-proteasome system (UPS) and autophagy system are the main proteolytic systems in cells, in order to distinguish the systems that may be involved in cPLA2 protein degradation, we used the proteasome inhibitor MG132, lysosome inhibitor chloroquine (CQ), quinacrine or CHX to treat GBM cell lines for 24 hours. Figure 1As shown in F: When cPLA2 was treated with CHX alone, cPLA2 was degraded in large quantities. MG132 could reverse this effect, but CQ could not. Compared with CHX monotherapy, dual treatment with quinacrine and CHX further enhanced the degradation of cPLA2. However, both MG132 and CQ could reverse this effect. These results indicate that under normal conditions, cPLA2 is only degraded by the UPS, while quinacrine further induces cPLA2 degradation through autophagy, which means that quinacrine has a new regulatory effect on the net balance of cPLA2 protein. At the same time, the above results suggest that p62 may mediate the autophagic degradation of cPLA2, but the possibility that the UPS is not involved in the accelerated degradation of cPLA2 cannot be ruled out. In order to verify the above hypothesis, an immunoprecipitation experiment was performed using an anti-cPLA2 antibody. After quinacrine treatment of GBM cells for 8 hours, the cell pellets were collected to prevent the massive degradation of cPLA2. As shown in Figure 1 G, Figure 1 Figure H: Under the action of quinacrine, the ubiquitination level of cPLA2 remains unchanged, but the binding of cPLA2 to p62 is significantly increased, indicating that cPLA2 is rapidly loaded into autophagosomes for degradation. To further verify that the decreased stability of cPLA2 is due to p62-mediated selective autophagy, we used small interfering RNA to knock down p62 in GBM (KD) and treated GBM cells with CHX or CHX + quinacrine for 24 hours ( Figure 1 I). The results showed that p62 knockdown did not affect the protein level of cPLA2 compared with the control group, which also means that pre-existing p62 does not mediate the autophagic degradation of cPLA2. As expected: in the p62 knockdown group, no additional cPLA2 degradation was observed after combination therapy compared with CHX monotherapy, as shown in Figure 1. Figure 5 These results indicate that increased p62 binding is crucial for the selective autophagic degradation of cPLA2 following quinacrine treatment.

[0065] Example 2: Quinacrine synergistically inhibits GBM proliferation with linagliptin

[0066] To further explore the anti-tumor effect of quinacrine combined with linagliptin, we first tested the effect of quinacrine alone on GBM proliferation. CCK8 results showed that quinacrine can inhibit tumor proliferation in multiple GBM cell lines ( Figure 2 A). Synergistic effect analysis results showed that quinacrine can produce synergistic effects with linagliptin in multiple GBM cell lines. Compared with the monotherapy group, the combination therapy can further inhibit GBM proliferation ( Figure 2 B- Figure 2 D).

[0067] Example 3: Quinacrine combined with linagliptin damages mitochondria and inhibits cellular energy metabolism, and the effect is better than AACOCF3 combined with linagliptin

[0068] We further compared the tumor inhibition effects of quinacrine combined with linagliptin and AACOCF3 combined with linagliptin. We found that low-concentration linagliptin combined with quinacrine can achieve the same effect as high-concentration linagliptin combined with AACOCF3 ( Figure 3 A). We also found that both quinacrine combined with linagliptin and AACOCF3 combined with linagliptin downregulated mitochondrial respiratory chain-related marker proteins ( Figure 3 B). Moreover, the effect of quinacrine combined with linagliptin was better than that of AACOCF3 combined with linagliptin. We also found that the damage effect of quinacrine combined with linagliptin on mitochondria was better than that of AACOCF3 combined with linagliptin. The evaluation indicators included mean branch length, mean perimeter and mean area ( Figure 3 C, Figure 3 D). Mitochondrial stress test and glycolysis stress test also showed similar results. Quinacrine combined with linagliptin inhibited basal respiration, ATP production, glycolysis and glycolysis capacity better than AACOCF3 combined with linagliptin. Figure 3 E- Figure 3 H).

[0069] Example 4: Quinacrine combined with linagliptin inhibits glioblastoma proliferation in vivo

[0070] To further explore the tumor-suppressing effect of quinacrine combined with linagliptin in vivo, we injected CT2A cells into the hippocampus of C57 mice to construct an intracranial glioma xenograft model ( Figure 4 A) and DMSO, quinacrine (10 mg / kg), and linagliptin (5 mg / kg) were administered orally. The effects of quinacrine combined with linagliptin on the tumor immune microenvironment were also further examined. Bioluminescence analysis showed that although both quinacrine and linagliptin alone inhibited tumor growth compared to DMSO, the quinacrine combined with linagliptin treatment group had the greatest effect on reducing tumor burden among the four treatment groups ( Figure 4 B- Figure 4 C). In addition, the survival curves showed the same results: the combination therapy group had the highest overall survival benefit compared with the single drug treatment group ( Figure 4 D- Figure 4 E). We further tested the levels of perforin (PF1) and human granzyme B (Gzms-B) and found that both quinacrine and linagliptin alone could increase their levels, but the degree of increase was lower than that in the combined treatment group ( Figure 4 F- Figure 4 G). The results of iNOS and CD8α immunofluorescence in tumor tissues showed that the infiltration of inflammatory cells in the tumor of the combined treatment group was the highest ( Figure 4 H- Figure 4 J). The above results show that the combined effect of quinacrine and linagliptin on tumor inhibition in vivo is superior to that of the monotherapy group, and promotes the maximum infiltration of immune cells.

[0071] Example 5: Quinacrine combined with linagliptin promotes mitochondrial-lysosomal contact

[0072] Mitochondria-lysosome contact sites (MLCS) are a hot topic in recent cell biology research. This dynamic membrane contact plays a key role in various cellular functions, including mitochondrial quality control. In implementation case 3, we also found that quinacrine combined with linagliptin downregulated mitochondrial respiratory chain-related marker proteins ( Figure 3 B). We then used super-resolution imaging to examine the contact between mitochondria and lysosomes under the action of quinacrine and linagliptin. The results showed that quinacrine combined with linagliptin significantly increased the contact time and the proportion of long-term contact between mitochondria and lysosomes, and the intensity was higher than that of AACOCF3 combined with linagliptin ( Figure 5 A- Figure 5 C). We then transferred TOM20-Venus and LAMP1-mTurquoise2 into glioma cells and detected lysosomal lifespan by fluorescence resonance energy transfer (FRET). Figure 5 D). The results showed that quinacrine combined with linagliptin significantly reduced the fluorescence intensity and lysosomal lifespan of lysosomes and promoted the colocalization of mitochondria and lysosomes ( Figure 5 E- Figure 5 H)

[0073] Example 6: Quinacrine combined with Linagliptin promotes mitochondrial-related protein degradation and apoptosis

[0074] Based on the above mechanism, we treated GBM cells with the protein synthesis inhibitor cycloheximide (CHX) or CHX combined with quinacrine and linagliptin in a time-dependent manner. Immunoblotting results showed that the degradation rate of mitochondrial-related proteins in the combination group was faster than that in the single drug group ( Figure 6 A- Figure 6B). Moreover, immunoblotting results related to PINK1 and Parkin showed that the combination of quinacrine and linagliptin had no significant effect on the protein levels of either, thus ruling out the role of mitochondrial autophagy ( Figure 6 C), which further indicates that the degradation of mitochondrial-related proteins is due to the contact between mitochondria and lysosomes. Transmission electron microscopy results also showed that under the action of quinacrine and linagliptin, the degradation of mitochondria increased, and the average length and number were significantly reduced ( Figure 6 D- Figure 6 F). Apoptosis analysis results also showed that the combination of quinacrine and linagliptin significantly promoted the mitochondrial-related apoptosis pathway, thereby inhibiting GBM proliferation ( Figure 6 G).

[0075] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of quinacrine combined with linagliptin in the preparation of drugs for treating glioma.

2. A pharmaceutical composition for treating glioma, characterized in that: include: Preparations containing quinacrine and preparations containing linagliptin.

3. The pharmaceutical composition for treating glioma according to claim 2, characterized in that: The mass ratio of quinacrine to linagliptin in the pharmaceutical composition is 2:

1. Preferably, the daily dosage of quinacrine is 10 mg / kg body weight, and the daily dosage of linagliptin is 5 mg / kg body weight.

4. The pharmaceutical composition for treating glioma according to claim 2, characterized in that: The pharmaceutical composition also includes pharmaceutically acceptable excipients.

5. The pharmaceutical composition for treating glioma according to claim 3, characterized in that: The excipients include but are not limited to diluents, fillers, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, fillers, flavorings, preservatives, dispersants, film formers, plasticizers, pore formers, pH regulators, opacifiers, retardants, and solvents.

6. The pharmaceutical composition for treating glioma according to claim 2, characterized in that: The dosage forms of the preparation containing quinacrine and the preparation containing linagliptin include but are not limited to capsules, tablets, powders, injections, and oral preparations.

7. Use of a drug combining quinacrine and linagliptin in the preparation of a drug for treating glioma.

8. The use according to claim 6, characterized in that: The drug changes the metabolic state of tumor cells, thereby increasing the sensitivity of tumor cells to linagliptin and enhancing the therapeutic effect.

9. Use of quinacrine in the preparation of a sensitizer for linagliptin in the treatment of glioma.

10. The use according to claim 9, characterized in that: The sensitizer increases the sensitivity of tumor cells to linagliptin by changing the metabolic state of tumor cells, thereby enhancing the therapeutic effect.

Images