WO2021179494A1 - 一种预防和/或治疗脑瘤的药物及其应用 - Google Patents

一种预防和/或治疗脑瘤的药物及其应用 Download PDF

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WO2021179494A1
WO2021179494A1 PCT/CN2020/099658 CN2020099658W WO2021179494A1 WO 2021179494 A1 WO2021179494 A1 WO 2021179494A1 CN 2020099658 W CN2020099658 W CN 2020099658W WO 2021179494 A1 WO2021179494 A1 WO 2021179494A1
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plasmodium
drug
brain tumors
tumor
brain
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PCT/CN2020/099658
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French (fr)
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陶铸
丁文婷
程智鹏
冯银芳
秦莉
陈小平
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广州中科蓝华生物科技有限公司
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Priority to AU2020435555A priority Critical patent/AU2020435555A1/en
Priority to US17/910,763 priority patent/US20230110635A1/en
Publication of WO2021179494A1 publication Critical patent/WO2021179494A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • This application belongs to the field of biomedicine technology, and specifically relates to a medicine for preventing and/or treating brain tumors and its application.
  • Brain tumors are common clinical tumors, which seriously threaten human health and survival. Brain tumors are divided into primary brain tumors and metastatic brain tumors. Metastatic brain tumors are more common than primary brain tumors. Among metastatic brain tumors, lung cancer is the most common, followed by breast cancer and melanoma, accounting for 67-80% of the total brain metastases. The most common primary brain tumors are glioma, meningioma, pituitary adenoma, and nerve sheath tumor, among which glioma accounts for 40% to 50% of primary tumors, and glioma in adults It accounts for 75% of malignant primary brain tumors.
  • gliomas are classified into grade 1 (the lowest degree of malignancy and the best prognosis) to grade 4 (the highest degree of malignancy and the worst prognosis).
  • grade 1 the lowest degree of malignancy and the best prognosis
  • grade 4 the highest degree of malignancy and the worst prognosis.
  • Glioblastoma corresponds to grade 4 as malignant.
  • Glioma has a median survival time of only 14.6 months.
  • brain tumors are unique. Due to their location in the brain and the presence of the blood-brain barrier, the traditional three major tumor treatment strategies are surgical resection and radiotherapy. Both chemotherapy and chemotherapy have certain limitations, which not only affect the performance of the therapy, but also cause serious side effects, which seriously affect the treatment of brain tumors. Among them, surgical treatment is to remove the brain tumor as cleanly as possible, but also to ensure that the brain function is not damaged. For the extremely difficult type of malignant glioma, the tumor shows infiltrating growth, and the boundary between normal brain tissue is not obvious. Surgery is difficult to completely remove, and it is easy to recur.
  • the existing immunotherapies such as targeted therapy, immune checkpoint inhibitors, and other clinical trials have unsatisfactory results and cannot improve survival. Therefore, it is very meaningful to develop a new treatment strategy that can obtain a significant anti-brain tumor effect, and it can also provide a new brain tumor treatment idea.
  • This application provides a medicine for preventing and/or treating brain tumors and applications thereof.
  • the drug can significantly inhibit the growth of brain tumors, significantly prolong the survival period of patients, break through the blood-brain barrier to exert curative effects, and has no obvious side effects and high safety. It provides new strategies and ideas for the treatment of brain tumors.
  • the present application provides a drug for preventing and/or treating brain tumors, the drug includes Plasmodium.
  • the drugs involved in this application break through the blood-brain barrier by fully activating the immune system.
  • the danger signal molecules released by malaria parasites are pathogen-related pattern recognition molecules (PAMPs), including known glycosylphosphatidylinositol anchors (GPI anchors), malaria pigments, malaria parasite DNA, immunostimulatory nucleic acid motifs and other unknown molecules, It can be recognized by the pattern recognition receptors (PRRs) of the host's immune cells.
  • PRRs include toll-like receptors (TLRs) on the surface of endosomal or cell membranes, RIG-I-like receptors (RLR) and NOD-like receptors (NLR) in the cytoplasm.
  • PRRs activated by Plasmodium PAMPs trigger different transcriptional programs And stimulate multiple downstream signaling pathways to induce a systemic immune response, release Th1 type cytokines such as TNF- ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-12, type I and type II IFNs, and activate NK cells, NKT cells, ⁇ / ⁇ T cells, macrophages and dendritic cells (DC), then deactivate CD4+ and CD8+ T cells.
  • Th1 type cytokines such as TNF- ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-12, type I and type II IFNs
  • NK cells NK cells
  • NKT cells ⁇ / ⁇ T cells
  • macrophages and dendritic cells DC
  • DAMPs Plasmodium infection damage related molecular patterns
  • meningeal lymphatics, peri-cerebrovascular spaces, prelymphatic lymphatics and perineural lymphatics play a vital role in the lymphatic circulation of the central nervous system. Plasmodium activates the immune system through the above pathways to fully activate the immune system and break through the blood-brain barrier.
  • malaria parasites can easily control the infection rate of malaria parasites through antimalarial drugs to control toxic side effects, clinically, malaria parasite infections are controllable and safe. This drug provides new ideas for the prevention and/or treatment of brain tumors. Ideas.
  • the brain tumor includes a primary brain tumor or a metastatic brain tumor.
  • the brain tumor includes glioma, meningioma, pituitary adenoma or nerve sheath tumor, preferably glioma.
  • the drugs involved in the present application have good effects on the prevention and/or treatment of brain tumors, especially on gliomas, and have anti-brain tumor effects by inducing a strong anti-tumor immune response.
  • the Plasmodium includes any one or a combination of at least two of Plasmodium falciparum, Plasmodium vivax, Plasmodium vivax, Plasmodium ovale, or Plasmodium nori.
  • the combination of at least two such as the combination of Plasmodium falciparum and Plasmodium vivax, the combination of Plasmodium vivax and Plasmodium ovale, the combination of Plasmodium falciparum and Plasmodium vivax, etc., any other combination is acceptable. The choices will not be repeated here. Preference is given to Plasmodium vivax and Plasmodium falciparum.
  • the plasmodium is a plasmodium after cryopreservation and/or resuscitation. In another preferred embodiment, the plasmodium is a plasmodium sporozoite after cryopreservation and/or resuscitation.
  • Cryopreservation quality control Take an appropriate amount of the RBC-glycerol cryopreservation solution mixture before cryopreservation and add an appropriate amount of RPMI 1640 medium. After incubating at 37°C in a CO 2 incubator for 72 hours, observe the color of the medium to confirm that there is no turbidity.
  • Preliminary preparations monitor the protozoan density and gametophyte status of volunteers infected with malaria parasites, collect 2 mL of peripheral blood intravenously, 24 hours in advance, and fast for Anopheles mosquitoes (above 300);
  • cryopreservation and resuscitation methods involved in the present application are more conducive to malaria parasite infection, improve infection efficiency, and improve the effectiveness of malaria parasite in preventing and/or treating brain tumors.
  • the dosage form of the drug is any pharmaceutically acceptable dosage form.
  • any pharmaceutically acceptable dosage form for example, tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or pills, etc.
  • the dosage form of the drug is injection.
  • the medicament further includes any one or a combination of at least two of the pharmaceutically acceptable pharmaceutical excipients.
  • the drugs involved in this application can be administered alone or in combination with adjuvants to form an appropriate dosage form for administration.
  • the adjuvants include diluents, thickeners, excipients, flavoring agents, fillers, binders, and lubricants. Any one or a combination of at least two of wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, pH regulators, antioxidants, bacteriostatic agents, or buffers .
  • the combination of at least two such as a combination of a diluent and an excipient, a combination of an emulsifier and a co-solvent, a combination of a filler and a binder and a wetting agent, and the like.
  • the drug is a drug loaded on a pharmaceutical carrier.
  • the pharmaceutical carrier includes liposomes, micelles, dendrimers, microspheres or microcapsules.
  • the application provides the application of the above-mentioned medicament in the preparation of a medicament for the prevention and/or treatment of brain tumors.
  • the present application provides a method for treating brain tumors with the above-mentioned drugs.
  • the specific method of treatment includes the following steps: using the above-mentioned drugs on patients with brain tumors, and the method of use only needs to input the drugs into the brain.
  • a person skilled in the art can select a method known in the art to carry out the successful infection of the tumor patient with the malaria parasite according to the actual situation, preferably the injection method.
  • Plasmodium infection can be through the bite of the natural vector female Anopheles mosquitoes or through blood transfusion of blood containing the plasmodium or the use of a syringe containing blood of the plasmodium.
  • the application provides a method of treating brain tumors, including:
  • step (b) includes: maintaining the Plasmodium infection for 6-8 weeks.
  • the brain tumor includes a primary brain tumor or a metastatic brain tumor.
  • the brain tumor includes glioma, meningioma, pituitary adenoma or nerve sheath tumor, preferably glioma.
  • the method further includes: (c) administering an antimalarial drug to terminate the malaria parasite infection.
  • steps (a), (b) and (c) are performed once.
  • steps (a), (b), and (c) are performed multiple times, resulting in repeated Plasmodium infections.
  • the Plasmodium may be the same or different.
  • the number of malaria parasites that can infect patients with brain tumors is feasible. According to individual differences in patients, the number of malaria parasites that can be infected is different. Those skilled in the art can adjust the number of malaria parasites according to the actual situation.
  • the inoculation amount of malaria parasites described in this application is not less than 100 active malaria parasites. Infected red blood cells, or no less than 5 active sporozoites of Plasmodium.
  • the Plasmodium infection is a long-term Plasmodium infection.
  • the longer the infection cycle the more obvious the inhibitory effect on brain tumors.
  • the long-term Plasmodium infection means that the Plasmodium continues to the chronic phase of the Plasmodium infection or at least maintains it. In the subacute stage, and maintain it for a period of time, antimalarial drugs are given to terminate the infection. After the malaria parasite is infected, after about 6-8 weeks of acute and subacute infection, it enters the chronic phase. At this time, only a small amount of malaria parasites can be detected in the peripheral blood, but no clinical symptoms of the acute phase appear.
  • Plasmodium infection can be re-infected after cured, it can also be repeatedly infected by different species of Plasmodium, so it can form a state of repeated Plasmodium infection. Plasmodium infection can inhibit the growth of brain tumor cells and prevent the spread and metastasis of tumor cells. , Extend the lifespan of brain tumor patients, win longer treatment opportunities and the body's immune environment for brain tumor patients, and contribute to the long-term survival and recovery of brain tumor patients.
  • fever caused by malaria parasite infection may promote the death of brain tumor cells. Plasmodiumemia is necessary to effectively inhibit the growth of brain tumors. However, in mice, Plasmodium causes only short-term infections without fever. It is difficult to observe repeated Plasmodium infections in mouse models. In people who lack effective anti-malarial treatment, Plasmodium infection can cause long-term parasitemia, accompanied by acute high fever, this syndrome can be repeated many times throughout the life. Therefore, malaria parasites obtained through the bite of Anopheles mosquitoes or artificially injected sporozoites will cause malaria parasite infections in the hepatic and blood phases. Continuous stimulation of the immune system can transform brain tumors into effective brain tumor vaccines. Its fever and its tumor angiogenesis inhibitory effect play a multi-channel and multi-target therapeutic effect on brain tumors. In the medical literature, febrile infection is related to the spontaneous regression of tumors, and malaria is a typical febrile infection.
  • the drugs involved in this application have significant preventive and/or therapeutic effects on brain tumors, can inhibit the growth of brain tumors, and prolong the lifespan of patients;
  • Plasmodium immunotherapy is mainly for intermittent cold and heat attacks.
  • Antimalarial drugs can be used to control the infection rate and avoid severe cold and heat attacks. The safety is guaranteed. Endanger the normal function of the brain and the functions of other organs and tissues;
  • chemotherapeutic drugs cannot easily cross the blood-brain barrier and cannot exert the anti-tumor effect of chemotherapeutics; many targets Drugs cannot enter brain tumors to exert direct anti-tumor effects; while radiotherapy requires radiation to the brain; the drug of this application mainly regulates the body’s immune function and inflammation-related factors to inhibit the growth of brain tumors, thereby prolonging the lifespan of patients ;
  • the drugs involved in this application and the malaria parasite immunotherapy using them are relatively economical and have relatively few side effects. They only need simple symptomatic treatment, regular monitoring of blood routines and liver and kidney functions, and patients do not need additional costs. , And can be treated again or terminated at any time according to changes in the condition, and the course of treatment is artificially controllable; it can not only reduce the burden on the patient, but also integrate the advantages of immunotherapy, fever therapy and anti-angiogenesis therapy.
  • Figure 1 is a graph showing the effect of malaria parasite infection on the growth of glioma in tumor-bearing mice in a subcutaneous inoculation model
  • Figure 2 is a graph showing the effect of malaria parasite infection on the survival time of tumor-bearing mice in a subcutaneous vaccination model
  • Figure 3 is a graph showing the effect of malaria parasite infection on the body weight of tumor-bearing mice in a subcutaneous inoculation model
  • Figure 4 is a graph showing the change trend of the malaria parasite infection rate in the malaria parasite treatment group in the subcutaneous inoculation model
  • Figure 5 is a schematic diagram of pWPXLd-mcherry-F2A-Luciferase-Puro lentiviral vector
  • Figure 6 is a visual field view of the positive rate of GL261.mcherry-Luc cell line single clone
  • Figure 7 is a graph showing the statistical results of Luciferase enzyme activity in the GL261.mcherry-Luc cell line
  • Fig. 8 is a result of in vivo imaging of the growth of gliomas in tumor-bearing mice by Plasmodium infection in an intracranial orthotopic vaccination model;
  • Figure 9 is a graph showing the effect of malaria parasite infection on the growth of gliomas in tumor-bearing mice in an intracranial orthotopic vaccination model
  • Figure 10 is a graph showing the effect of malaria parasite infection on the survival time of tumor-bearing mice in an intracranial orthotopic vaccination model
  • Figure 11 is a graph showing the effect of malaria parasite infection on the body weight of tumor-bearing mice in an intracranial orthotopic vaccination model
  • Figure 12 is a graph showing the changing trend of the malaria parasite infection rate in the malaria parasite treatment group in the intracranial orthotopic vaccination model.
  • This embodiment provides a method for freezing and resuscitating blood of Plasmodium worms, and the specific operations are as follows:
  • the malaria parasite blood is quickly divided into packages as required, and the packed blood from the malaria parasites is labeled and stored in liquid nitrogen.
  • Plasmodium peripheral blood of malaria patients without other legal blood-borne infectious diseases, or blood of plasmodium cultured in the laboratory;
  • Reagents Sodium chloride injection, 10% NaCl solution, 3.5% NaCl solution, RPMI1640 medium, 28% glycerol cryopreservation solution (mix 28 g of glycerol, 3 g of sorbitol, and sodium chloride injection and dilute the volume to 100 mL volume);
  • cryopreservation tube 50mL centrifuge tube, 15mL centrifuge tube, 250mL culture bottle, pasteurizer, printing label and registration form;
  • Cryopreservation quality control take a proper amount of the RBC-28% glycerol cryopreservation solution mixture before cryopreservation and add a proper amount of 10% FBS-RPMI1640 medium, 37°C, CO 2 incubator after 72 hours, observe the color of the medium to confirm that there is no turbid;
  • Resuscitation quality control After the inoculation is completed, the remaining blood samples, blood smears, microscopic examination to observe the infection rate of the worms and the protozoan status before the injection, and make a record.
  • This embodiment provides a method for cryopreservation, resuscitation and inoculation of plasmodium sporozoites, and the specific operations are as follows:
  • Collect blood detect the density of malaria parasites and gametophytes in volunteers infected with malaria. Collect 2 mL of peripheral blood from the volunteers intravenously, put them in a thermos cup immediately, and transport them to the mosquito room within 2 hours;
  • Anopheles feeding blood Adjust the temperature of the mosquito room to 26°C before feeding blood, prepare the film blood feeding system, add the collected peripheral blood to the film blood feeding system, and feed the Anopheles blood for 30 minutes;
  • thermos Record the temperature before and after the transportation of the thermos, the time of feeding blood, the number of Anopheles mosquitoes, and the blood feeding situation of Anopheles mosquitoes, etc.;
  • Sporozoite inspection check the sporozoites on the 14th-16th day after the blood is infected, dissected 20 Anopheles mosquitoes, calculate the total sporozoites, and average the amount of spores per Anopheles mosquito;
  • This example explores the therapeutic effect of malaria parasite infection on tumor-bearing mice in a subcutaneous brain tumor inoculation model.
  • 10127101P-G purchased from Jiangsu Shitai Laboratory Equipment Co., Ltd.; Cedar oil: Shanghai Specimen Model Factory, China; PBS buffer: SH30256.01, purchased from Hyclone Company; Physiological saline: purchased from Chen Xin Pharmaceutical Co., Ltd.
  • mice On the first day after tumor inoculation, the mice were randomly divided into two groups according to their weight: the control glioma group (GL261), the Plasmodium yoelii treatment group (GL261+Py), with 10 mice in each group.
  • control glioma group GL261
  • Plasmodium yoelii treatment group GL261+Py
  • Plasmodium inoculation C57BL/6 mice were inoculated by intraperitoneal injection of 0.2 mL/mouse after mixing, and two mice were inoculated as breeding mice each time;
  • Tumor volume measurement The tumor is measured every 3 days, and the tumor volume (in cubic millimeters) is calculated using the ellipse volume calculation formula: (D ⁇ d ⁇ d)/2, where "D” represents the long diameter of the tumor, " d” means short diameter.
  • the tumor size was expressed as the average tumor volume ⁇ standard mean error (SEM), and the tumor growth curve was made. Perform statistical analysis between groups by TWO-WAY ANOVA analysis of variance. When p ⁇ 0.05, it is represented by "*”, and when p ⁇ 0.01, it is represented by "**”, both of which indicate that the difference between groups is of significant statistical significance;
  • Plasmodium infection rate is evaluated by the percentage of mouse red blood cells infected with Plasmodium. The calculation formula is: (number of erythrocytes infected with plasmodium/total erythrocytes) ⁇ 100%; the specific operation is to take blood from the tail vein The slices were fixed with methanol and stained with Giemsa stain. The number of Plasmodium infecting red blood cells and the total number of red blood cells were observed under a microscope. The total number of red blood cells was about 1000. The infection rate of Plasmodium was calculated. The infection rate was based on the average infection rate ⁇ standard average Error (SEM) representation, and draw the Plasmodium infection cycle curve to observe whether the chemotherapy drugs have an impact on the Plasmodium infection;
  • SEM standard average Error
  • the GL261/mcherry-luc cell line was constructed to prepare for the exploration of the therapeutic effect of malaria parasite infection on tumor-bearing mice in the brain tumor intracranial orthotopic vaccination model in Example 5.
  • Cells GL261, mouse glioma cell line; HEK293T cell line, used for lentivirus packaging;
  • Plasmid pWPXLd-mcherry-F2A-Luciferase-Puro; Transfection reagent: PEI, purchased from Polysciences; Bright-Glo TM luciferase detection system: Promega; Countstar FL automatic cell fluorescence analyzer: purchased from Shanghai Rui Yu Biotechnology Co., Ltd.; Endotoxin-free plasmid DNA mass extraction kit: N1051, purchased from Guangzhou Dongsheng Biotechnology Co., Ltd.; Puromycin: purchased from Invivogen Company; DMEM basic medium, 11995065, purchased from Gibco; FBS fetus Bovine serum, 04-001-1ACS, purchased from Biological Industries; Reduced Serum Medium, article number 31985-070, purchased from ThermoFisher.
  • PEI purchased from Polysciences
  • Bright-Glo TM luciferase detection system Promega
  • Countstar FL automatic cell fluorescence analyzer purchased from Shanghai Rui Yu Biotechnology Co., Ltd.
  • Plasmid extraction Refer to the instruction manual of endotoxin-free plasmid DNA mass extraction kit N1051.
  • This example explores the therapeutic effect of malaria parasite infection on tumor-bearing mice in a brain tumor intracranial orthotopic vaccination model.
  • In vivo imager IVIS Spectrum, purchased from PerkinElmer; Brain stereotaxic instrument: purchased from Shenzhen Reward Life Technology Co., Ltd.; Mini handheld cranial drill: purchased from Shenzhen Reward Life Technology Co., Ltd.; Micro syringe: 5 ⁇ L Specifications, purchased from Hamilton; electronic digital oil gauge calipers: article number 678040, purchased from pioneering tools; surgical instruments (surgical pads, timers, scalpel blades, scalpel handles, ophthalmic surgical scissors, ophthalmic surgical straight tweezers, ophthalmic surgical curved tweezers , Needle holder, stitches, cotton swabs, sutures).
  • surgical instruments surgical pads, timers, scalpel blades, scalpel handles, ophthalmic surgical scissors, ophthalmic surgical straight tweezers, ophthalmic surgical curved tweezers , Needle holder, stitches, cotton swabs, sutures).
  • Cell resuscitation resuscitate the mouse glioma cell line GL261/mcherry-luc and culture it in a 5% CO 2 , 37°C constant temperature incubator;
  • Inject cells set the injection needle at the bregma position, use X, Y, and Z digital displays to determine the injection position (right 1.8mm, front 1mm, depth 3.4mm), inject cells, injection speed of 1 ⁇ L About 1 min, keep the needle for 5 min, and withdraw the needle slowly when withdrawing the needle;
  • mice are in vivo imaging, and the total luminous flux of the tumor on the head of the mouse is calculated;
  • Plasmodium resuscitation The blood of Plasmodium murine (1.0mL/branch) frozen in a liquid nitrogen tank is quickly shaken in a 37°C water bath to mix and melt to keep the Plasmodium active;
  • each mouse On the 7th day after tumor subcutaneous inoculation, each mouse will be inoculated with 0.2mL, that is, each mouse will be inoculated with 5 ⁇ 10 5 Plasmodium, and the control group will be inoculated with the same number of red blood cells without Plasmodium. liquid.
  • In vivo imaging of mice Perform in vivo imaging of mice every 7 days to calculate the total luminous flux of the brain to characterize the size of the brain tumor.
  • the tumor size is expressed as the average tumor volume ⁇ standard mean error (SEM), and the tumor growth curve is made .
  • Plasmodium infection rate is evaluated by the percentage of mouse red blood cells infected with Plasmodium. The calculation formula is: (number of erythrocytes infected with plasmodium/total erythrocytes) ⁇ 100%; the specific operation is to take blood from the tail vein The slices were fixed with methanol and stained with Giemsa stain. The number of Plasmodium infecting red blood cells and the total number of red blood cells were observed under a microscope. The total number of red blood cells was about 1000. The infection rate of Plasmodium was calculated. The infection rate was based on the average infection rate ⁇ standard average Error (SEM) representation, and draw the Plasmodium infection cycle curve to observe whether the chemotherapy drugs have an impact on the Plasmodium infection;
  • SEM standard average Error

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Abstract

一种预防和/或治疗脑瘤的药物及其应用，所述药物包括疟原虫。该药物对脑瘤具有显著的预防和/或治疗效果，能够抑制脑瘤的生长，延长患者的寿命；且无明显的毒副作用，安全性高；该药物丰富了脑瘤的治疗手段，由于脑部组织的特殊性和敏感性，化疗药物不容易穿过血脑屏障，不能发挥抗肿瘤效应，而该药物主要通过调节机体的免疫功能以及炎症相关因子，通过全面激活免疫能力突破血脑屏障，抑制脑瘤的生长，从而延长寿命。

Description

一种预防和/或治疗脑瘤的药物及其应用 技术领域
本申请属于生物医药技术领域，具体涉及一种预防和/或治疗脑瘤的药物及其应用。
背景技术
脑瘤是临床上常见的肿瘤，严重威胁人类的健康和生存。脑瘤分为原发性脑瘤和转移性脑瘤两种，转移性的脑肿瘤比原发性的脑肿瘤更常见。在转移性脑瘤中，以肺癌最为常见，其次是乳腺癌和黑色素瘤，约占总的脑转移瘤的67-80％。在原发性脑肿瘤中最多见的依次为胶质瘤、脑膜瘤、垂体腺瘤和神经鞘膜瘤，其中胶质瘤占原发性肿瘤的40％～50％，在成人中胶质瘤占了75％的恶性原发性脑瘤。根据WHO制定的分级系统，将脑胶质瘤分为1级(恶性程度最低、预后最好)到4级(恶性程度最高、预后最差)，胶质母细胞瘤与4级相对应属于恶性胶质瘤，只有14.6个月的中位生存期。
脑瘤与其他神经系统的癌症、肺癌、肝癌、乳腺癌等其他癌症相比具有特殊性，由于其发病部位在脑部，以及血脑屏障的存在，传统三大肿瘤治疗策略即手术切除、放疗和化疗都有一定的局限性，不但影响疗法的发挥，而且还会导致严重的毒副作用，严重影响了脑瘤的治疗。其中，手术治疗是尽可能把脑瘤切除干净，还要保证不损伤大脑功能，而对于恶性胶质瘤这种极难以治愈的类型，肿瘤呈现浸润性生长，与正常脑组织的界限不明显，手术难以完全切除干净，很容易复发，还可能存在脑部术后并发症，如流血、血凝块和肿胀等，对于脑干神经胶质瘤和大脑深部位置的脑瘤，手术治疗的风险更大，目前最常用的治疗手段是手术切除治疗配合放化疗。但是放疗可能破坏正常脑组织的损伤和坏死，还存在诱发新的肿瘤和中风等风险；而某些化疗药物虽然可以通过改善血脑屏障的通透性从而提高药效，但会引起瘤旁水肿、癫痫、认知障碍和抑郁等副作用。因此，脑瘤的预防和治疗一直是一个世界科学难题。
其中，胶质母细胞瘤患者的5年期生存只有3％，肿瘤的免疫疗法的不断发展为脑瘤提供了高特异性和低毒副作用的治疗策略，免疫疗法现在也被广泛用于研究治疗脑瘤试验，可以增强抗肿瘤的免疫能力，但是对于恶性的胶质瘤，还未达到改善生存质量的预期。许多早期临床试验结果表明，胶质瘤的疫苗虽然可能取得一定治疗的效果，但在随机对照试验中却不能改善生存。免疫检查点抑制剂治疗胶质母细胞瘤的临床试验也显示对患者的生存没有显著影响，胶质母细胞瘤对免疫检查点抑制剂没有反应。因此脑瘤的免疫疗法还需要进一步研究。
由于传统的三大疗法对脑瘤的治疗效果有限，现有的免疫疗法，如靶向治疗、免疫检查点抑制剂等临床试验结果不理想，不能改善生存。因此，开发出一种新的能获得显著抗脑瘤效果的治疗策略是非常有意义的，也可以提供一种新的脑瘤治疗思路。
发明内容
本申请提供了一种预防和/或治疗脑瘤的药物及其应用。该药物能够显著抑制脑瘤的生长，并显著延长患者的生存期，突破血脑屏障发挥疗效，且无明显的毒副作用，安全性高，为脑瘤的治疗提供了新的策略和思路。
一方面，本申请提供了一种预防和/或治疗脑瘤的药物，所述药物包括疟原虫。
本申请所涉及的药物通过全面激活免疫系统突破血脑屏障。疟原虫释放的危险信号分子病原体相关的模式识别分子(PAMPs)，包括已知的糖基磷脂酰肌醇锚(GPI锚)、疟色素、疟原虫DNA、免疫刺激核酸基序和其他未知分子，能被宿主的免疫细胞的模式识别受体(PRRs)识别。PRRs包括在内体膜或细胞膜表面的toll样受体(TLRs)，细胞质中的RIG-I样受体(RLR)和NOD样受体(NLR)，疟原虫PAMPs激活的PRRs触发不同的转录程序和刺激多个下游信号通路诱导产生全身免疫反应，释放Th1型细胞因子如TNF-α，IL-1β，IL-2，IL-6，IL-12，I型和II型IFNs，激活NK细胞，NKT细胞，γ/δT细胞，巨噬细胞和树突状细胞(DC)，然后再去激活CD4+和CD8+T细胞。通过下调肿瘤组织中的TGF-β，IL-10等免疫抑制因子，从而显著减少肿瘤内部的调节性T细胞(Tregs)、骨髓来源抑制性细胞(MDSCs)和肿瘤相关巨噬细胞(TAMs)等的数量，从而使肿瘤内部的免疫抑制微环境重塑为免疫支持的微环境，最终可以将脑瘤转化为有效的脑瘤疫苗。另一方面，疟原虫感染损伤相关分子模式(DAMPs)，如内源性尿酸、微泡和疟原虫感染的红细胞也诱导相似的免疫活性。研究表明，脑膜淋巴管、脑血管周围间隙、淋巴管前淋巴系统及神经周围淋巴管在中枢神经系统淋巴循环中起到了至关重要的作用，疟原虫通过以上途径全面激活免疫系统突破血脑屏障，实现抑制脑瘤生长，达到预防和/或治疗脑瘤的功效。因为疟原虫可以通过抗疟药很容易地控制疟原虫的感染率从而控制毒副作用，在临床上疟原虫感染是可控的、安全的，该药物为脑瘤的预防和/或治疗提供了新的思路。
在一个实施方案中，所述脑瘤包括原发性脑瘤或转移性脑瘤。
在一个优选的实施方案中，所述脑瘤包括胶质瘤、脑膜瘤、垂体腺瘤或神经鞘膜瘤，优选胶质瘤。
本申请所涉及的药物对预防和/或治疗脑瘤具有很好的效果，尤其对胶质瘤具有很好的效果，其通过诱导强烈的抗肿瘤免疫反应起到抗脑瘤的效应。
在另一个实施方案中，所述疟原虫包括恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫或诺氏疟原虫中的任意一种或至少两种的组合。所述至少两种的组合例如恶性疟原虫和间日疟原虫的组合、三日疟原虫和卵形疟原虫的组合、恶性疟原虫和间日疟原虫的组合等，其他任意的组合方式均可选择，在此便不再一一赘述。优选间日疟原虫和恶性疟原虫。
在一个优选的实施方案中，所述疟原虫为经过冻存和/或复苏后的疟原虫。在另一个优选的实施方案中，所述疟原虫为经过冻存和/或复苏后的疟原虫子孢 子。
所述冻存复苏疟原虫包括如下具体步骤：
(1)疟原虫冻存
(1.1)含疟原虫全血经300×g离心5min，血浆转至另一50mL离心管中；
(1.2)吸去白细胞层，加入2倍体积1640培养基，混匀，300×g离心5min；
(1.3)将等体积的28％甘油冻存液溶液缓慢滴加到50mL离心管中，边加边震荡混匀，室温孵育5min；
(1.4)将RBC-甘油冻存溶液混合物，每管1.0mL分装于冻存管中；
(1.5)放入冻存盒中，直接放入液氮保存；
(1.6)冻存质量控制：取适量冻存前的RBC-甘油冻存液溶液混合物加入适量的RPMI 1640培养基，37℃，CO 2培养箱培养72h后，观察培养基颜色，确认无浑浊。
(2)疟原虫复苏
(2.1)准备：水浴锅(37℃)、50mL离心管、3.5％NaCl溶液、氯化钠注射液、离心机、打印标签、表格；
(2.2)检查核对冻存管标签；
(2.3)取冻存的疟原虫血，于37℃中水浴1-3min，快速混匀，解冻；
(2.4)转移至15mL离心管中，沿着管壁缓慢加入等体积的3.5％NaCl溶液，轻轻吹打混匀，室温静置5min，弃上清；
(2.5)沿管壁缓慢加入5倍体积的0.9％NaCL溶液，轻轻吹打混合均匀，300×g离心5min，弃上清；
(2.6)加入生理盐水至红细胞压积为50％，混匀，计数并记录红细胞密度，暂存于4℃，用于临床接种；
(2.7)疟原虫复苏质量控制：接种完成后，剩余血样，血涂片，镜检观察注射前疟原虫的感染率和原虫状况，并做好记录。
所述冻存复苏疟原虫子孢子包括如下具体步骤：
(1)饲血按蚊
(1.1)前期准备：监控疟原虫感染的志愿者原虫密度与配子体情况，静脉采集外周血2mL，提前24h，按蚊禁食(300只以上)；
(1.2)饲血前，调整蚊房温度至26℃，准备好薄膜饲血系统，将采集的外周血，添加至薄膜死穴系统中，饲血30min；
(1.3)吸去未吸饱按蚊，写好标签，将按蚊放入26℃培养箱饲养，添加含10％葡萄糖+0.05％PABA的糖水棉。
(2)卵囊检查
(2.1)饲血感染后第7-10天检查卵囊，解剖10只，计算阳性按蚊比例；注意事项：卵囊感染标准，+：1-10个；++：11-100个；+++：101个及以上；
(2.2)如果卵囊感染度“++”以上比例＜50％，复查20只按蚊。
(3)子孢子检查
(3.1)饲血感染后第14-16天检查子孢子，解剖20只按蚊，计算子孢子总量，平均每只按蚊子孢子的量。注意事项：计数用细胞计数板，统计大方格内所有子孢子数目，并遵循“数上不数下，数左不数右”的原则；为尽量减少误差，计数时，计数三次取平均值。
(4)子孢子获取
(4.1)饲血感染后第14-20天，取出按蚊，用75％乙醇消毒按蚊3次，每次5s，然后用昆虫生理盐水冲洗按蚊3次，每次5s。组织技术人员解剖唾液腺，收集于装有200μL的AB+人血清的EP管中，EP管始终置于冰上；注意事项：如果直接接种，解剖好的唾液腺放入生理盐水即可。获得按蚊唾液腺，用18G针头注射器反复吹打30-40次，破碎唾液腺和组织，1000rpm，4℃，离心5min，收集含子孢子的上清液；
(4.2)注意事项：解剖时尽量取到按蚊完整的唾液腺，避免把组织碎片一起收集，否则会影响唾液腺的破碎和子孢子的纯化；子孢子获取是按照虫株分别进行，做好标记，防止子孢子获取过程中出现虫株间交叉污染；获取子孢子的过程应控制在1h内进行；
(5)子孢子冻存
(5.1)上述含子孢子的上清液，取少许，血球计数板计数子孢子；
(5.2)12000rpm，4℃，离心10min，弃上清，加适量含1％三抗的AB型人血清重悬沉淀，调整子孢子浓度至2.5×10 8/mL；
(5.3)200μl/管分装至冻存管中，做好标记后投放入-80℃酒精中速冻，3h后转移至液氮罐中。
(6)子孢子直接接种
(6.1)准备1mL注射器、75％酒精、棉签；
(6.2)获得的子孢子生理盐水悬液，无菌操作，取少许，血球计数板计数子孢子，调整子孢子浓度至接种数量；
(6.3)静脉注射，接种，隔离和观察；
(6.4)留取少量接种前样本进行无菌试验，并做好记录；
(6.5)接种后3天，每天血涂片，镜检疟原虫。
(7)子孢子复苏后接种
(7.1)准备1mL注射器、75％酒精、棉签；
(7.2)开启紫外消毒30min以上；
(7.3)从液氮罐中取出保存的子孢子，在37℃水浴锅中解冻1-2min；
(7.4)12000rpm，4℃离心10min，弃上清，加入10倍体积生理盐水重悬；
(7.5)12000rpm，4℃离心10min，弃上清，加入200μL生理盐水重悬；
(7.6)静脉注射，接种，隔离和观察；
(7.7)留取少量接种前样本进行无菌试验，并做好记录；
(7.8)接种后3天，每天血涂片，镜检疟原虫。
本申请所涉及的上述冻存和复苏的方法更有利于疟原虫进行感染，提高感染效率，提高疟原虫在预防和/或治疗脑瘤中的效果。
在另一个实施方案中，所述药物的剂型为药剂学上可接受的任意一种剂型。例如片剂、散剂、混悬剂、颗粒剂、胶囊剂、注射剂、喷雾剂、溶液剂、灌肠剂、乳剂、膜剂、栓剂、贴剂、滴鼻剂或滴丸剂等。
在一个优选的实施方案中，所述药物的剂型为注射剂。
在另一个实施方案中，所述药物还包括药剂学上可接受的药用辅料中的任意一种或至少两种的组合。
本申请所涉及的药物可单独给药也可以与辅料搭配做成适当的剂型进行给药，所述辅料包括稀释剂、增稠剂、赋形剂、调味剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。所述至少两种的组合例如稀释剂和赋形剂的组合、乳化剂和助溶剂的组合、填充剂和粘合剂和润湿剂的组合等。
在另一个实施方案中，所述药物为负载于药用载体上的药物。
在一个优选的实施方案中，所述药用载体包括脂质体、胶束、树枝状大分子、微球或微囊。
另一方面，本申请提供了如上所述的药物在制备预防和/或治疗脑瘤的药物中的应用。
再一方面，本申请提供一种利用如上所述药物治疗脑瘤的方法，治疗的具体方法包括如下步骤：对脑瘤患者使用如上所述的药物，所述使用方法只需要将药物输入到脑瘤患者体内使其成功感染疟原虫，本领域技术人员可以根据实际情况选择本领域公知的方法进行，优选为注射的方式。疟原虫感染可以通过自然传播媒介雌性按蚊叮咬人或者通过血液输入带有疟原虫的血液或使用含有疟原虫的血液的注射器等。
在一个实施方案中，本申请提供了一种治疗脑瘤的方法，包括：
(a)向患者施用治疗有效量的本申请所提供的药物；以及
(b)使疟原虫感染维持至慢性期或亚急性期。
在一个优选的实施方案中，步骤(b)包括：使疟原虫感染维持6-8周。
在一个实施方案中，所述脑瘤包括原发性脑瘤或转移性脑瘤。
在一个实施方案中，所述脑瘤包括胶质瘤、脑膜瘤、垂体腺瘤或神经鞘膜瘤，优选胶质瘤。
在一个实施方案中，该方法还包括：(c)施用抗疟药终止疟原虫感染。
在一个实施方案中，步骤(a)、(b)和(c)进行一次。
在另一个实施方案中，步骤(a)、(b)和(c)进行多次，从而形成反复疟原虫感染。在该实施方案的反复疟原虫感染中，疟原虫可以相同或不同。
在本申请中，能让脑瘤患者感染疟原虫的疟原虫数目都是可行的。根据患者个体差异，能感染上疟原虫的数目有所不同，本领域技术人员可以根据实际情况调节疟原虫的数目，本申请所述疟原虫的接种量为不低于100个有活性的疟原虫感染的红细胞，或不低于5个有活性的疟原虫子孢子。
在本申请中，所述疟原虫感染为长程疟原虫感染，感染周期越长，对脑瘤的抑制效果越明显，所述长程疟原虫感染为疟原虫持续到疟原虫感染的慢性期或至少维持到亚急性期，并维持一段时间，再给予抗疟药终止感染。疟原虫感染后经过约6-8周的感染急性期和亚急性期后，进入慢性期，此时仅能在外周血中检测到少量的疟原虫，但不出现急性期的临床症状。
由于人疟原虫感染治愈后可以再感染，也可以被不同种的疟原虫反复感染，所以可以形成反复的疟原虫感染状态，疟原虫感染能够抑制脑瘤细胞的生长，防止肿瘤细胞的扩散和转移，延长脑瘤患者的寿命，为脑瘤患者赢得更长的治疗时机和机体的免疫环境，有助于脑瘤患者的长期生存和康复。
在本申请中，疟原虫感染引起的发热可能促进脑瘤细胞的死亡。疟原虫血症是有效抑制脑瘤生长所必需的，然而在小鼠中，疟原虫仅引起短期感染而没有发烧，在鼠模型中难以观察到重复的疟原虫感染。在缺乏有效抗疟疾治疗的人中，疟原虫感染可引起长期寄生虫血症，伴随急性期高热，这种综合征可在整个生命期重复许多次。因此，通过按蚊叮咬自然获得的或人工方法注射子孢子获得的疟原虫将产生肝期和血期的疟原虫感染，连续刺激免疫系统可以将脑瘤转变为有效的脑瘤疫苗，与急性期的发热及其肿瘤血管生成抑制效应等对脑瘤起到多通路多靶点的治疗作用。在医学文献中，发热性感染与肿瘤的自发消退有关，疟疾是典型的发热性感染。
相对于现有技术，本申请具有以下有益效果：
(1)本申请所涉及的药物对脑瘤具有显著的预防和/或治疗效果，能够抑制脑瘤的生长，延长患者的寿命；
(2)本申请所涉及的药物的毒副作用有限，安全性高；疟原虫免疫疗法主要是间歇性寒热发作，可以使用抗疟药控制感染率，避免剧烈的寒热发作，安全性有保证，不危害脑部正常功能和其他器官组织的功能；
(3)本申请所涉及的药物丰富了脑瘤的治疗手段，由于脑部组织的特殊性和敏感性，化疗药物不容易穿过血脑屏障，不能发挥化疗药物的抗肿瘤效应；很多靶向药物也不能进入脑部肿瘤发挥直接抗肿瘤效应；而放疗需要对脑部进行辐射；本申请药物主要通过调节机体的免疫功能，以及炎症相关的因子，抑制脑瘤的生长，从而延长患者的寿命；
(4)长程的疟原虫感染对脑瘤的治疗效果更好，且本申请所涉及的疟原虫 免疫疗法治疗脑瘤时不以发热时间作为疗程标准，而应在保护患者的脏器功能和生命安全的前提下，延长疟原虫感染时间直至能控制肿瘤的进展；
(5)本申请所涉及的药物及利用其进行的疟原虫免疫疗法相对经济，且副作用相对较少，仅需简单的对症治疗、定期监测血常规及肝肾功能，患者不需额外的其他费用，而且可以依据病情变化，随时再行治疗或终止治疗，疗程人为可控；既能减轻患者负担，又综合了免疫疗法、发热疗法和抗血管生成疗法的优势。
附图说明
图1是皮下接种模型中疟原虫感染对荷瘤小鼠胶质瘤生长的影响结果图；
图2是皮下接种模型中疟原虫感染对荷瘤小鼠的生存时间的影响结果图；
图3是皮下接种模型中疟原虫感染对荷瘤小鼠的体重的影响结果图；
图4是皮下接种模型中疟原虫治疗组的疟原虫感染率的变化趋势图；
图5是pWPXLd-mcherry-F2A-Luciferase-Puro慢病毒载体示意图；
图6是对GL261.mcherry-Luc细胞系单克隆进行阳性率检测的视野图；
图7是GL261.mcherry-Luc细胞系Luciferase酶活性统计结果图；
图8是颅内原位接种模型中疟原虫感染对荷瘤小鼠的胶质瘤生长的活体成像结果图；
图9是颅内原位接种模型中疟原虫感染对荷瘤小鼠的胶质瘤生长的影响结果图；
图10是颅内原位接种模型中疟原虫感染对荷瘤小鼠生存时间影响结果图；
图11是颅内原位接种模型中疟原虫感染对荷瘤小鼠的体重的影响结果图；
图12是颅内原位接种模型中疟原虫治疗组的疟原虫感染率的变化趋势图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例1
本实施例提供一种疟原虫虫血的冻存与复苏方法，具体操作如下：
感染疟原虫的血液入库后，快速按要求将疟原虫血进行分装，分装好的疟原虫虫血贴上标签，液氮冻存。
(一)疟原虫冻存：
(1)实验材料准备：
疟原虫：无其它法定血源传染病的疟疾患者外周血，或实验室培养的疟原虫虫血；
试剂：氯化钠注射液、10％的NaCl溶液、3.5％的NaCl溶液、RPMI1640培养基、28％甘油冻存溶液(将甘油28g、山梨醇3g、氯化钠注射液混合后定 容至100mL体积)；
耗材：冻存管、50mL离心管、15mL离心管、250mL培养基瓶、巴氏管、打印标签和登记表格；
仪器：移液器、移液枪、灭菌剪刀、液氮罐；
(2)采血与镜检：取适量虫血样本，制作薄血片，进行镜检，冻存的为30％以上为早期滋养体时期疟原虫，填写镜检报告；
(3)每管20mL虫血，300×g离心10min(升降速都设置1)，血浆转移至新的50mL离心管中；
(4)吸去离心管底部上层的白细胞层，加入2倍虫血体积的1640培养基，混匀，300×g离心5min(升降速都设置1)；
(5)轻弹50mL离心管管底，将与虫血等体积的28％甘油冻存溶液缓慢滴加到50mL离心管中，用移液器轻轻吹打均匀，室温孵育5min；
(6)将红细胞(RBC)-28％甘油冻存溶液混合物，每管1.0mL体积分装于冻存管中，贴好打印的标签，放入冻存盒中，直接放入液氮冻存；
(7)冻存质量控制：取适量冻存前的RBC-28％甘油冻存溶液混合物加适量10％FBS-RPMI1640培养基，37℃，CO 2培养箱培养72h后，观察培养基颜色确认无浑浊；
(二)疟原虫复苏：
(1)试剂与耗材准备：水浴锅(37℃)、15mL离心管、3.5％NaCl、氯化钠注射液、离心机、打印标签、表格；
(2)取冻存的虫血(1.0mL/支)，于37℃水浴锅快速振荡，快速解冻，保持疟原虫活性；
(3)将虫血加入到15mL离心管中，沿管壁缓慢加入等体积的3.5％NaCl，轻轻吹打混匀，室温静置5min，300×g离心5min，弃上清；
(4)沿管壁缓慢加入5倍虫血体积的0.9％NaCl注射液，轻轻吹打混匀，300×g离心5min，弃上清；
(5)加生理盐水至红细胞压积为50％，混匀，计数并记录红细胞密度，暂存于4℃，用于临床接种；
(6)复苏质量控制：接种完成后，剩余血样，血涂片，镜检观察一下注射前虫子的感染率和原虫状况，并做好记录。
实施例2
本实施例提供一种疟原虫子孢子的冻存、复苏与接种方法，具体操作如下：
(一)按蚊饲血与子孢子获取
(1)按蚊准备：提前24h，饥饿300只以上按蚊；
(2)采集虫血：检测疟疾感染的志愿者疟原虫密度与配子体情况，静脉采集志愿者的外周血2mL，立即放入保温杯中，2h内运输至蚊房；
(3)按蚊饲血：饲血前调整蚊房温度至26℃，准备好薄膜饲血系统，将采集的外周血添加至薄膜饲血系统中，按蚊饲血30min；
(4)按蚊饲养：吸去未吸饱的按蚊，写好标签，将按蚊放入26℃培养箱饲养，添加含10％葡萄糖+0.05％PABA的糖水棉；
(5)记录保温杯运输前后的温度、饲血时间、按蚊数量、按蚊饲血情况等；
(6)卵囊检查：饲血感染后第7-10天检查卵囊，解剖10只，计算阳性蚊子比例。卵囊感染度标准，+：1-10个，++：11-100个，+++：101个及以上；如果卵囊感染度“++”以上比率＜50％，复查20只按蚊；
(7)子孢子检查：饲血感染后第14-16天检查子孢子，解剖20只按蚊，计算子孢子总量，平均每只按蚊子孢子的量；
(8)子孢子获取：饲血感染后第14-20天，取出按蚊，用75％乙醇消毒按蚊3次，5s/次，然后用昆虫生理盐水冲洗按蚊3次，5s/次。解剖唾液腺，收集于装有200mL的AB+人血清的EP管中，EP管始终置于冰上。如果直接接种，解剖好的唾液腺放入生理盐水即可；
(9)获得按蚊唾液腺，用18G针头注射器反复吸打30-40次，破碎唾液腺和组织，1000rpm，4℃，离心5min，收集含子孢子的上清液。解剖时尽量取到按蚊完整的唾液腺，避免把组织碎片一起收集，否则会影响唾液腺的破碎和子孢子的纯化；子孢子获取最好不同虫株分开进行，做好标记，防止子孢子获取过程中出现不同虫株间交叉污染；获取子孢子的过程控制在1h以内；
(二)子孢子冻存
(1)上述含子孢子的上清液，取少许，血球计数板计数子孢子；
(2)12000rpm，4℃，离心10min，弃上清，加适量含1％三抗的AB型人血清重悬沉淀，调整子孢子浓度至2.5×10 8/mL；
(3)200μL/管分装至冻存管中，做好标记后投入-80℃酒精中速冻，3h后转移至液氮罐中；
(三)子孢子直接接种
(1)获得的子孢子生理盐水悬液，无菌操作，取少许，血球计数板计数子孢子，调整子孢子浓度至接种数量；
(2)静脉注射，进行接种，并进行隔离观察；留取少量接种前样本进行无菌试验，并做好记录；接种后3天，每天血涂片，镜检疟原虫；
(四)子孢子复苏后接种
(1)从液氮罐中取出保存的子孢子，在37℃水浴锅中解冻1-2min；
(2)12000rpm，4℃，离心10min，弃上清，加10倍体积的生理盐水重悬；
(3)12000rpm，4℃，离心10min，弃上清，加200μL生理盐水重悬；
(4)静脉注射，进行接种，并隔离观察；
(5)留取少量接种前样本进行无菌试验，并做好记录；
(6)接种后3天，每天血涂片，镜检疟原虫。
实施例3
本实施例探究皮下脑瘤接种模型中疟原虫感染对荷瘤小鼠的治疗效果。
(一)实验材料和试剂：
动物：C57BL/6小鼠，雌性，6-8周龄，来源于上海斯莱克实验动物有限责任公司；疟原虫：小鼠约氏疟原虫(P.yoelii 17XNL，MRA-593，Py)，来自Malaria Research and Reference Reagent Resource Center(MR4)免费馈赠；显微镜：奥林巴斯显微镜CX31；游标卡尺：电子数显油标卡尺，货号678040，购自开拓工具；Giemsa染剂粉：购自Sigma-Aldrich公司；病理级载玻片：货号10127101P-G，购自江苏世泰实验器材有限公司；香柏油：中国上海标本模型厂；PBS缓冲液：SH30256.01，购自Hyclone公司；生理盐水：购自辰欣药业股份有限公司。
(二)实验步骤
(1)建立动物模型，具体方法为：
(1.1)细胞复苏：复苏小鼠胶质瘤细胞系GL261，在5％CO 2、37℃恒温培养箱中静置培养；
(1.2)细胞扩培：每2-3天传代一次，当细胞长满培养皿底部的80％时，用0.25％胰酶-EDTA消化液消化，按照1:3稀释传代；
(1.3)小鼠剃毛：C57BL/6小鼠右侧肩胛到右后背部剃毛干净；
(1.4)单细胞悬液制备：取对数生长期的细胞，经0.25％胰酶-EDTA消化液消化后，用PBS洗涤3次，用无血清1640培养基重悬细胞，细胞浓度为5×10 6；
(1.5)小鼠皮下肿瘤细胞接种：C57BL/6小鼠小鼠右侧肩胛区皮下接种，每只注射细胞悬液体积为0.1mL，GL261细胞接种量为5×10 5个/只，接种当天为肿瘤接种第0天；
(1.6)实验分组：肿瘤接种后第1天，按照体重大小随机分为两组：对照胶质瘤组(GL261)，约氏疟原虫治疗组(GL261+Py)，每组小鼠10只。
(2)疟原虫接种荷瘤小鼠，具体方法为：
(2.1)疟原虫复苏：将冻存于液氮罐中的鼠疟原虫血(1.0mL/支)，于37℃水浴锅中快速震荡，使其快速混匀融化，保持疟原虫活性；
(2.2)疟原虫接种：混匀后0.2mL/只腹腔注射接种C57BL/6小鼠，每次接种两只小鼠作为种鼠；
(2.3)薄血膜制作和镜检：小鼠剪尾采血取约1-1.5μL，涂于载玻片，制作2.5cm长舌状薄血膜，吹风机吹干。甲醇浸润血膜1min，1×Giemsa染液染色30min，用自来水冲洗干净，吹干。用油镜100×观察疟原虫感染率。观察疟原虫感染率变化；
(2.4)疟原虫溶液制备：当感染率达到3％-10％时，红细胞计数(一只感染疟原虫的种鼠，一只未接种疟原虫的
Figure PCTCN2020099658-appb-000001
小鼠)，剪尾巴取血5μL重悬于 995μL PBS中，红细胞计数，计算出每mL体积感染疟原虫的红细胞数。用0.2mL体积的3.8％枸橼酸钠抗凝剂润湿EP管，摘眼球采血，计算所需要的接种疟原虫的浓度和总量，用PBS配制浓度2.5×10 6/mL；
(2.5)接种荷瘤小鼠：肿瘤皮下接种后第1天，每只小鼠接种0.2mL，约氏疟原虫治疗组每只小鼠接种含5×10 5个疟原虫的红细胞液，对照组接种相同数量不含疟原虫的红细胞液。
(3)检测指标
(3.1)肿瘤体积大小测量：每3天进行肿瘤测量，用椭圆体积计算公式计算肿瘤体积(单位立方毫米)：(D×d×d)/2，其中“D”表示肿瘤的长径，“d”表示短径。肿瘤大小以平均肿瘤体积±标准平均误差(SEM)表示，制作肿瘤生长曲线。通过TWO-WAY ANOVA方差分析进行组间统计分析，当p≤0.05时，用“*”表示，当p≤0.01时，用“**”表示，均说明组间差异具有显著的统计学意义；
(3.2)小鼠生存统计：存活以中位生存期和延长的存活期的百分数来评定，采用Kaplan-Meier法估计生存率，做生存曲线图，计算中位生存时间。当p≤0.05时，用“*”表示，当p≤0.01时，用“**”表示，均说明组间差异具有显著的统计学意义；
(3.3)疟原虫感染率统计：疟原虫感染率以小鼠红细胞感染疟原虫百分数来评定，计算公式为：(感染疟原虫红细胞数目/总红细胞)×100％；具体操作为尾静脉取血涂片，用甲醇固定，并用吉姆萨染液染色，显微镜下观察感染红细胞的疟原虫数量和总红细胞数，总红细胞数在1000个左右，计算疟原虫感染率，感染率以平均感染率±标准平均误差(SEM)表示，并绘制疟原虫感染周期曲线，观察化疗药物是否对疟原虫感染有影响；
(3.4)小鼠体重称重：每3天进行称量，体重生长以平均体重±标准平均误差(SEM)来表示，观察疟原虫感染对荷瘤小鼠体重的影响。
(4)实验结果
(4.1)肿瘤生长曲线如图1所示，疟原虫感染治疗组与未感染的对照组比较，疟原虫感染显著抑制胶质瘤GL261的生长(p＝0.0244，*)；
(4.2)荷瘤小鼠生存曲线如图2所示，疟原虫感染治疗组与未感染的对照组比较，疟原虫感染显著延长荷瘤小鼠的生存期(p＝0.0479，*)，中位生存期从35.5天延长到41.5天；
(4.3)荷瘤小鼠体重的变化情况如图3所示，疟原虫感染率变化情况如图4所示，随着疟原虫感染上升，荷瘤小鼠体重会逐渐下降，在感染率最高时体重最低，随后随着疟原虫感染逐渐降低，小鼠体重逐渐上升。
实施例4
本实施例构建GL261/mcherry-luc细胞系，为实施例5中探究脑瘤颅内原位接种模型中疟原虫感染对荷瘤小鼠的治疗效果做准备。
(一)实验材料和试剂：
细胞：GL261，小鼠胶质瘤细胞系；HEK293T细胞系，用于慢病毒包装；
质粒：pWPXLd-mcherry-F2A-Luciferase-Puro；转染试剂：PEI，购买自Polysciences公司；Bright-Glo TM萤光素酶检测系统：Promega公司；Countstar FL全自动细胞荧光分析仪：购自上海睿钰生物科技有限公司；无内毒素质粒DNA大量提取试剂盒：N1051，购自广州东盛生物科技有限公司；嘌呤霉素：购自Invivogen公司；DMEM basic培养基，11995065，购自Gibco；FBS胎牛血清，04-001-1ACS，购自Biological Industries；
Figure PCTCN2020099658-appb-000002
Reduced Serum Medium，货号31985-070，购自ThermoFisher公司。
(二)实验步骤：
(1)慢病毒载体质粒
(1.1)构建pWPXLd-mcherry-F2A-Luciferase-Puro质粒，如图5所示；
(1.2)质粒抽提：使用方法参照无内毒素质粒DNA大量提取试剂盒N1051的使用说明书。
(2)慢病毒包装
(2.1)HEK-293T细胞培养：细胞处于指数生长状态；
(2.2)细胞消化：在转染前一天把细胞用胰酶消化下来，离心后用新鲜的完全培养基重悬传代于10cm培养皿中，5％CO2的37℃培养箱中，使细胞贴壁融合度达到80％以上；
(2.3)转染前2h更换新鲜的完全培养基；
(2.4)将转染所需的PEI、各个质粒、Opti-MEM I Reduced Serum Medium等放置室温平衡；
(2.5)制备质粒的混合液：先根据下表用量加入三种质粒，和Opti-MEM I Reduced Serum Medium，吹打均匀后室温放置10min；
Figure PCTCN2020099658-appb-000003
(2.6)再分别取PEI转染试剂120μL和Opti-MEM I Reduced Serum Medium1mL，混匀后室温放置10min；
(2.7)最后将以上溶液轻轻混合，混匀后室温放置10min，即为转染用的混合液；
(2.8)混合液逐滴加入细胞表面，混合均匀；
(2.9)转染6h后，更换培养基；
(2.10)转染48和72h后，收集上清；
(2.11)收集的病毒上清3000rpm离心30min；
(2.12)将离心后的病毒液再用0.45μm过滤器过滤，滤液保存于-80℃备用。
(3)构建GL261-luc-mcherry细胞系
(3.1)复苏GL261细胞系，并传代培养，使细胞处于对数生长时期；
(3.2)T25细胞培养瓶培养GL261细胞，用0.25％胰酶-EDTA消化成单细胞悬液；
(3.3)细胞计数，取5×10 5个细胞铺入6孔板中，加入培养基至1.8mL终体积；
(3.4)向6孔板中每孔细胞加入2μL Polybrene并摇匀；
(3.5)向6孔板中每孔细胞加入600μL pWPXLd-mCherry-F2A-Luciferase病毒上清液并摇匀，置于37℃培养箱中培养；
(3.6)转染24h后，弃去转染液，更换为正常培养基继续培养；
(3.7)转染48h后，弃去转染液，更换为2μg/ml浓度嘌呤霉素的筛选培养基继续培养；
(3.8)观察转染细胞的mCherry表达情况。
(4)GL261/mcherry-luciferase单克隆筛选
(4.1)转染72h后，将GL261/mCherry-Luc细胞消化下来并计数；
(4.2)取适量细胞悬液稀释，嘌呤霉素浓度为2μg/mL，在10cm细胞培养皿中加入50-100个细胞，摇匀后置于37℃培养；
(4.3)第二天在显微镜下标记出单个阳性细胞，每隔两天半换液；
(4.4)筛选18天后，将在显微镜下观察形态正常而且表达mCherry的克隆挑取至6孔板中扩大培养。
(5)GL261/mcherry-luciferase单克隆细胞系鉴定
(5.1)Count Star计数仪计算阳性率：将细胞样本充分吹散，混匀，吸取20μL加入计数板，静置10-30s，设置好参数，自动计数；
(5.2)将Bright-GloTM缓冲液加入Bright-Glo底物中，颠倒混合均匀，直到所有底物完全溶解，室温下放置平衡至室温；
(5.3)细胞计数，调整细胞浓度，1×10 6细胞/mL，96孔板每孔板加入100μL，即含1×10 5细胞，再加入100μL混合的Bright-Glo检测试剂；
(5.4)室温孵育2min，直到细胞裂解完全；
(5.5)使用多功能酶标仪进行化学发光检测。
(6)实验结果
(6.1)GL261.mcherry-Luc细胞系单克隆的阳性率检测视野图如图6所示：计算得到单克隆阳性率达到96.49％，可以认为是单克隆细胞系；
(6.2)GL261.mcherry-Luc细胞系Luciferase酶活性结果统计图如图7所示(图中con为对照组)：计算得到GL261/mcherry-luciferase-C1单克隆的荧光素 酶活性很强，可以用于小鼠体内活体成像实验。
实施例5
本实施例探究脑瘤颅内原位接种模型中疟原虫感染对荷瘤小鼠的治疗效果。
(一)实验材料和试剂
动物：C57BL/6小鼠，雌性，6-8周龄，来源于上海斯莱克实验动物有限责任公司；肿瘤细胞：GL261/mcherry-Luciferase，自己构建，见实施例4；疟原虫：小鼠约氏疟原虫(P.yoelii 17XNL，MRA-593，Py)，来自Malaria Research and Reference Reagent Resource Center(MR4)免费馈赠；荧光素酶底物：D-Luciferin，Potassium Salt D-荧光素钾盐，购买自上海翊圣生物科技有限公司；Giemsa染剂粉：购自Sigma-Aldrich公司；DMEM basic培养基，11995065，购自Gibco；FBS胎牛血清，04-001-1ACS，购自Biological Industries；阿佛丁(麻醉剂，自己配制)、酒精、碘酒、双氧水、生理盐水、硅油、硫酸庆大霉素注射液、红霉素软膏。
(二)实验仪器
活体成像仪：IVIS Spectrum，购自PerkinElmer公司；脑立体定位仪：购自深圳市瑞沃德生命科技有限公司；微型手持颅钻：购自深圳市瑞沃德生命科技有限公司；微量注射器：5μL规格，购自Hamilton；电子数显油标卡尺：货号678040，购自开拓工具；手术器械(手术垫、计时器、手术刀片、手术刀柄、眼科手术剪刀、眼科手术直镊子、眼科手术弯镊子、持针器、缝针、棉签、缝合线)。
(三)实验步骤
(1)建立动物模型
(1.1)细胞复苏：复苏小鼠胶质瘤细胞系GL261/mcherry-luc，在5％CO 2、37℃恒温培养箱中静置培养；
(1.2)细胞扩培：每2-3天传代一次，当细胞长满培养皿底部的80％时，用0.25％胰酶-EDTA消化液消化，按照1:3稀释传代；
(1.3)GL261/mcherry-luc细胞准备：取指数生长期的GL261/mcherry-luc细胞，PBS洗涤，胰酶消化，PBS洗涤3次，细胞计数，RPIM 1640培养基重悬，细胞浓度为2×10 8/μL，冰上保存；
(1.4)75％酒精消毒手术器械；
(1.5)准备注射针和微量注射器：用1mL注射器将进样针和连接软管充满硅油，检查有无堵管，防止有空气泡，将注射器更换为微量注射器，安装进样针，吸取适量的细胞量进行调试是否漏气；
(1.6)小鼠麻醉：阿佛丁麻醉剂按照0.4mL/20g体重的剂量腹腔给药，使得小鼠麻醉；
(1.7)小鼠剃毛：用剃毛刀将头顶毛发剃干净，尽量将眼睛到两耳间的毛 剃除干净；
(1.8)将小鼠固定到脑立体定位仪上，先固定小鼠牙齿(将门齿放在鼻夹的口子上)，用镊子稍微打开嘴巴，拉出舌头，然后固定头后部(将耳杆放在两耳中，主要用于固定颧骨)，最后固定紧鼻夹；
(1.9)用碘酒消毒皮肤，沿中线用手术刀切开皮肤，切口从眼后到耳朵约10mm，用手术刀辅助，刮头骨上的结缔组织，用棉签沾上双氧水擦拭头骨，使颅骨前囟更清晰；
(1.10)将进样针尖端对准前囱，以前囱为基准点，小鼠头部横向为X轴，纵向中线为Y轴，设置定位仪的X和Y轴为0mm，移动进样针，X轴前1mm为钻孔地方，Y轴中线旁右1.8mm，再下降进样针，对准小鼠头盖骨表面，再设置Z轴为0mm，上升进样针，用铅笔标记颅骨钻孔地方，孔径约2mm；
(1.11)注射细胞：再将进样针定在前囟位置，利用X、Y、Z数显仪定注射位置(右1.8mm，前1mm，深3.4mm)，注射细胞，1μL注射量注射速度约1min，留针5min，退针时需缓慢退针；
(1.12)缝合：缝合后用红霉素软膏涂抹伤口，再肌肉注射0.1mL硫酸庆大霉素注射液；
(1.13)活体成像：肿瘤接种第7天，小鼠活体成像，计算小鼠头部肿瘤部位的总光通量；
(1.14)实验分组：利用分层随机抽样分组，分为两组：对照胶质瘤组(GL261)，约氏疟原虫治疗组(GL261+Py)。
(2)疟原虫接种荷瘤小鼠，具体方法为：
(2.1)疟原虫复苏：将冻存于液氮罐中的鼠疟原虫血(1.0mL/支)，于37℃水浴锅中快速震荡，使其混匀融化，保持疟原虫活性；
(2.2)疟原虫接种：混匀后0.2mL/只腹腔注射接种C57BL/6小鼠，每次接种两只小鼠；
(2.3)薄血膜制作和镜检：小鼠剪尾采血取约1-1.5μL，涂于载玻片，制作2.5cm长舌状薄血膜，吹风机吹干。甲醇浸润血膜1min，1×Giemsa染液染色30min，用自来水冲洗干净，吹干。用油镜100×观察疟原虫感染率。观察疟原虫感染率变化；
(2.4)疟原虫溶液制备：当感染率达到3％-10％时，先红细胞计数，剪尾巴取血5μL重悬于995μL PBS中，红细胞计数。计算出每mL体积感染疟原虫的红细胞数。用0.2mL体积的3.8％枸橼酸钠抗凝剂润湿EP管，摘眼球采血，计算所需要的接种疟原虫的浓度和总量，用PBS配制浓度2.5×10 6/mL；
(2.5)接种荷瘤小鼠：肿瘤皮下接种后第7天，每只小鼠接种0.2mL，即每只小鼠接种5×10 5个疟原虫，对照组接种相同数量不含疟原虫的红细胞液。
(3)检测指标
(3.1)小鼠活体成像：每7天进行小鼠活体成像，计算脑部的总光通量， 来表征脑瘤的大小，肿瘤大小以平均肿瘤体积±标准平均误差(SEM)表示，制作肿瘤生长曲线。通过Two-Way ANOVA方差分析进行组间统计分析，当p≤0.05时，用“*”表示，当p≤0.01时，用“**”表示，均说明组间差异具有显著的统计学意义；
(3.2)小鼠生存统计：存活以中位生存期和延长的存活期的百分数来评定，采用Kaplan-Meier法估计生存率，制作生存曲线图，计算中位生存时间。当p≤0.05时，用“*”表示，当p≤0.01时，用“**”表示，均表明组间差异具有显著的统计学意义；
(3.3)疟原虫感染率统计：疟原虫感染率以小鼠红细胞感染疟原虫百分数来评定，计算公式为：(感染疟原虫红细胞数目/总红细胞)×100％；具体操作为尾静脉取血涂片，用甲醇固定，并用吉姆萨染液染色，显微镜下观察感染红细胞的疟原虫数量和总红细胞数，总红细胞数在1000个左右，计算疟原虫感染率，感染率以平均感染率±标准平均误差(SEM)表示，并绘制疟原虫感染周期曲线，观察化疗药物是否对疟原虫感染有影响；
(3.4)小鼠体重称重：每3天进行称量，体重生长以平均体重±标准平均误差(SEM)来表示，观察化疗药物和疟原虫感染对荷瘤小鼠体重的影响。
(4)实验结果
(4.1)疟原虫感染对荷瘤小鼠的胶质瘤生长的活体成像结果图如图8所示，肿瘤总光通量随时间的变化如图9所示，由图8和图9可知：疟原虫感染治疗组和对照组相比，疟原虫感染治疗显著抑制小鼠颅内原位接种的胶质瘤GL261的生长(p＝0.0003，**)；
(4.2)荷瘤小鼠生存曲线如图10所示，疟原虫感染治疗和对照组相比，疟原虫感染显著延长颅内原位接种的荷瘤小鼠的生存期，中位生存期从40.5天延长到55.5天(p＝0.0054，**)；
(4.3)荷瘤小鼠的体重变化图如图11所示，疟原虫感染率变化如图12所示，由图11和图12可知：对照组因肿瘤负荷加重，体重开始逐渐降低；疟原虫感染治疗组随着疟原虫感染上升，荷瘤小鼠体重会逐渐下降，在感染率最高时体重最低，随后随着疟原虫感染逐渐降低，小鼠体重逐渐上升。
申请人声明，本申请通过上述实施例来说明本申请的一种预防和/或治疗脑瘤的药物及其应用，但本申请并不局限于上述实施例，即不意味着本申请必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。
以上详细描述了本申请的优选实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征， 在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。

Claims (15)

  1. 一种预防和/或治疗脑瘤的药物，其包括疟原虫。
  2. 如权利要求1所述的预防和/或治疗脑瘤的药物，其中，所述脑瘤包括原发性脑瘤或转移性脑瘤。
  3. 如权利要求1所述的预防和/或治疗脑瘤的药物，其中，所述脑瘤包括胶质瘤、脑膜瘤、垂体腺瘤或神经鞘膜瘤，优选胶质瘤。
  4. 如权利要求1-3中任一项所述的预防和/或治疗脑瘤的药物，其中，所述疟原虫包括恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫或诺氏疟原虫中的任意一种或至少两种的组合，优选间日疟原虫和恶性疟原虫。
  5. 如权利要求1-4中任一项所述的预防和/或治疗脑瘤的药物，其中，所述疟原虫为经过冻存和/或复苏后的疟原虫或者经过冻存和/或复苏后的疟原虫子孢子。
  6. 如权利要求1-5中任一项所述的预防和/或治疗脑瘤的药物，其中，所述药物的剂型为药剂学上可接受的任意一种剂型。
  7. 如权利要求1-6中任一项所述的预防和/或治疗脑瘤的药物，其中，所述药物的剂型为注射剂。
  8. 如权利要求1-7中任一项所述的预防和/或治疗脑瘤的药物，其中，所述药物还包括药剂学上可接受的药用辅料中的任意一种或至少两种的组合；
    优选地，所述药用辅料包括增稠剂、稀释剂、调味剂、粘合剂或填充剂中的任意一种或至少两种的组合。
  9. 如权利要求1-8中任一项所述的预防和/或治疗脑瘤的药物，其中，所述药物为负载于药用载体上的药物；
    优选地，所述药用载体包括脂质体、胶束、树枝状大分子、微球或微囊。
  10. 一种治疗脑瘤的方法，包括：
    (a)向患者施用治疗有效量的权利要求1-9中任一项所述的药物；以及
    (b)使疟原虫感染维持至慢性期或亚急性期。
  11. 如权利要求10所述的法，其中步骤(b)为：使疟原虫感染维持6-8周。
  12. 如权利要求10或11所述的方法，其中所述脑瘤包括原发性脑瘤或转移性脑瘤。
  13. 如权利要求12所述的方法，其中，所述脑瘤包括胶质瘤、脑膜瘤、垂体腺瘤或神经鞘膜瘤，优选胶质瘤。
  14. 如权利要求10-13中任一项所述的方法，其还包括：(c)施用抗疟药终止疟原虫感染；并且
    其中，步骤(a)、(b)和(c)进行一次或更多次。
  15. 如权利要求1-9中任一项所述的药物在制备预防和/或治疗脑瘤的药物中的应用。
PCT/CN2020/099658 2020-03-13 2020-07-01 一种预防和/或治疗脑瘤的药物及其应用 WO2021179494A1 (zh)

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