WO2015192663A1 - 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用 - Google Patents

重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用 Download PDF

Info

Publication number
WO2015192663A1
WO2015192663A1 PCT/CN2015/071893 CN2015071893W WO2015192663A1 WO 2015192663 A1 WO2015192663 A1 WO 2015192663A1 CN 2015071893 W CN2015071893 W CN 2015071893W WO 2015192663 A1 WO2015192663 A1 WO 2015192663A1
Authority
WO
WIPO (PCT)
Prior art keywords
rlz
lung cancer
tumor
cancer
glioma
Prior art date
Application number
PCT/CN2015/071893
Other languages
English (en)
French (fr)
Inventor
张喜田
孙非
梁重阳
Original Assignee
张喜田
孙非
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 张喜田, 孙非 filed Critical 张喜田
Publication of WO2015192663A1 publication Critical patent/WO2015192663A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the invention belongs to the field of biomedicine and relates to the application of recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) in the preparation of anti-tumor drugs, and relates to recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) in anti-lung cancer, laryngeal cancer and glial
  • rLZ-8 recombinant Ganoderma lucidum immunoregulatory protein
  • the therapeutic effect of tumor growth improves the living condition of tumor-bearing mice and improves the survival rate of tumor-bearing mice.
  • Lung cancer is a malignant tumor derived from the epithelial cells of the bronchi or bronchioles, accounting for 90-95% of malignant tumors of the lung parenchyma. Lung cancer is the most common primary malignancy in the lungs. According to the World Health Organization survey, the incidence of lung cancer accounts for the first place in malignant tumors in many countries and regions. The disease is more than 40 years old, the peak age of onset is between 60-79 years old. The prevalence rate for men and women is 2.3:1. Lung cancer originates from the bronchial mucosa epithelium, and is confined to the basement membrane to become carcinoma in situ.
  • non-small cell lung cancer occurs more, and about 80% of lung cancer patients belong to this type.
  • patients with small cell lung cancer are mainly treated with chemotherapy, and surgical treatment does not play a major role in this type of lung cancer patients.
  • Surgical treatment is mainly for non-small cell lung Cancer patients.
  • the main cause of lung cancer is the increasing exposure to environmental risk factors, such as the widespread presence of smokers and passive smokers, the increase in respiratory diseases (tuberculosis, chronic obstructive pulmonary disease, asthma, etc.), the increase in urban air pollution, Incomplete treatment of kitchen fumes, reduced intake of fresh vegetables and fruits, etc.
  • respiratory diseases such as the widespread presence of smokers and passive smokers
  • respiratory diseases such as the widespread presence of smokers and passive smokers, the increase in respiratory diseases (tuberculosis, chronic obstructive pulmonary disease, asthma, etc.)
  • the increase in urban air pollution Incomplete treatment of kitchen fumes, reduced intake of fresh vegetables and fruits, etc.
  • Incomplete treatment of kitchen fumes Incomplete treatment of kitchen fumes
  • reduced intake of fresh vegetables and fruits etc.
  • Laryngocarcinoma is a type of cancer of the digestive system. Most laryngeal cancers are laryngeal squamous cell carcinoma (LSCC), which accounts for about 95%. Laryngeal cancer is mainly formed on squamous epithelial cells of the laryngeal epithelium. Laryngeal cancer can be formed at any location in the larynx, and the recovery rate of laryngeal cancer is greatly affected by the location of cancer cells. Laryngeal cancer can be divided into three basic types: glottic (vocal), glottic (sound) and subglottic.
  • glottic vocal
  • glottic sound
  • subglottic subglottic
  • Laryngeal cancer is most common with glottic type, followed by glottic type, and the least occurs is subglottic. According to statistics, laryngeal cancer accounts for 1.2-1.6% of systemic malignant tumors, accounting for 3.3-8.1% of head and neck malignant tumors. Laryngeal cancer has the characteristics of high morbidity and high recurrence rate. Males have a high incidence. The annual incidence of new diseases is 3.5-5.5/100,000, and that of females is 0.6/100,000. The incidence rate in developed countries is high, and the mortality rate is about 2.4/100,000. The female is 0.3/100,000. Among respiratory tumors, the incidence of laryngeal cancer ranks second.
  • the treatment of laryngeal cancer is mainly through the rational application of surgery, radiotherapy, chemotherapy and biological therapy to achieve the maximum elimination of tumors, preservation of laryngeal function and improvement of patient survival rate.
  • Surgical treatment is the preferred treatment for laryngeal cancer, but surgical treatment inevitably causes laryngeal damage and also affects the patient's quality of life.
  • Radiotherapy for laryngeal cancer is suitable for supraglottic laryngeal carcinoma with lesions >1 cm, superficial, and no metastasis, and the general condition is poor, and it is not suitable for surgery.
  • laryngeal cancer cells have different sensitivities to radiation, and some radiation-resistant cells survive even under high-intensity illumination.
  • Induction chemotherapy for laryngeal cancer plays an important role in organ retention. Current clinical trial results show that chemotherapy can improve the throat retention rate and reduce distant metastasis, and the overall survival rate of patients is not improved.
  • Glioma is a type of glioma.
  • a glioma is a tumor that begins in the brain or spine. It is called glioma because it is produced by glial cancer. The glioma is the most common.
  • Brain tumors are associated with primary and metastatic heterogeneous populations of the central nervous system and are life-threatening and low-sustaining diseases. The annual incidence of primary malignant brain tumors is approximately 24,000.
  • Malignant glioma is a tumor that occurs in the glial site and accounts for 70% of brain tumors.
  • the treatment methods of glioma mainly include surgery, radiotherapy and chemotherapy.
  • the glioma is characterized by diffuse and invasive infiltration into the surrounding brain tissue, and there is no obvious boundary with the surrounding tissue. Therefore, the tumor cannot be completely removed by surgery, and the tumor cells in the dormant phase may be activated to rapidly immerse into the proliferative phase. It is easy to relapse after resection, more than 2cm within the tumor bed, and the treatment effect is poor.
  • radiotherapy has a very limited effect. When the radiation dose reaches 73-80Gy, it can effectively kill glioma cells, while the normal brain tissue can tolerate only 60Gy, and some malignant brain tumors.
  • gliomas have certain resistance to chemotherapeutic drugs and most chemotherapeutic drugs for treating glioma have strong toxic side effects, chemotherapeutic drugs for gliomas have lower efficacy.
  • Ganoderma lucidum is a medicinal fungus that has been used as a medicinal history for thousands of years. It has been reported that Ganoderma lucidum has immunomodulatory and anti-tumor effects.
  • Ganoderma lucidum immunoregulatory protein (LZ-8) is a fungal immunoregulatory protein extracted from Ganoderma lucidum.
  • the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) provided in this paper is a redesigned code.
  • the LZ-8 gene was fully synthesized, and the obtained protein sample was recombinantly expressed and purified by the Pichia pastoris expression system. Previous studies have shown that rLZ-8 also has certain antitumor activity.
  • rLZ-8 can effectively kill human lung gland.
  • Tumor cell lines such as cancer tumor cells (A549) and human gastric adenocarcinoma cells (SGC7901), but the therapeutic effects of rLZ-8 on lung cancer, laryngeal cancer and glioma have not been reported.
  • the invention proposes that rLZ-8 has significant therapeutic effects on lung cancer, laryngeal cancer and glioma.
  • the invention relates to the application of rLZ-8 in the preparation of medicines for treating lung cancer, laryngeal cancer and glioma, and shows that rLZ-8 has significant inhibition on lung cancer, laryngeal cancer and glioma through a series of experimental methods and results.
  • the role of the specific invention is as follows:
  • a lung cancer metastasis model was established using healthy and flexible C57BL/6 mice.
  • the rLZ-8 in vivo anti-lung cancer experimental group was designed, including normal control group, model group, rLZ-8 low-dose group, rLZ-8 medium-dose group and rLZ-8 high-dose group, each group. See the example 1 for the dosage.
  • the survival time of the tumor-bearing mice was used as the investigation object, and the administration was 30 days respectively. After 60 days of administration, the number of lung nodules and survival of each group of mice were counted, and the tumor inhibition rate of tumor-bearing mice and the survival rate of tumor-bearing mice were calculated.
  • rLZ-8 in vivo anti-larynx cancer research designed four experimental groups including model control group (saline), rLZ-8 low-dose group, rLZ-8 medium-dose group, rLZ-8 high-dose group, among which The doses of the low, medium and high dose groups of rLZ-8 were 125 ⁇ g/kg, 250 ⁇ g/kg, and 500 ⁇ g/kg, respectively, and the administration time was 60 days and 90 days.
  • the therapeutic effect index mainly records the body weight, tumor weight, tumor volume and calculates the tumor inhibition rate based on the tumor weight.
  • Statistical analysis of the data showed that rLZ-8 was effective in the treatment of laryngeal cancer.
  • a subcutaneous xenograft model of glioma nude mice was established using healthy and flexible BALB/c-nude nude mice.
  • Four experimental groups including model control group (normal saline), rLZ-8 low dose group, rLZ-8 medium dose group and rLZ-8 high dose group were designed, among which rLZ-8 low, medium and high doses were used.
  • the doses of the group were 125 ⁇ g/kg, 250 ⁇ g/kg, and 500 ⁇ g/kg, respectively.
  • the days of administration were 60 days and 90 days.
  • the therapeutic effect index mainly records the body weight, tumor weight, tumor volume and calculates the tumor inhibition rate based on the tumor weight.
  • Statistical analysis of the data showed that rLZ-8 was effective in the treatment of glioma.
  • the invention has the beneficial effects that the invention uses the in vivo test method to verify that rLZ-8 is used for treating lung cancer.
  • the application of laryngeal cancer and glioma through a series of pharmacodynamic experiments showed that rLZ-8 has significant effects in the treatment of lung cancer, laryngeal cancer and glioma.
  • rLZ-8 not only improves the treatment of lung cancer.
  • the tumor inhibition rate prolongs the survival time of tumor-bearing mice, and a tumor-bearing mouse model with a certain proportion of lung cancer nodules disappears, which is unexpected by the inventors; and in the treatment of laryngeal cancer and glioma The same effect of subcutaneous solid tumor disappeared.
  • the concentration of rLZ-8 protein stock solution was 1 mg/ml, which was set to rLZ-8 low dose (250 ⁇ g/kg) 31.25 ⁇ g/ml solution, rLZ-8 medium dose (500 ⁇ g/kg) 62.5 ⁇ g/ml solution, which was rLZ- 8 high dose (1000 ⁇ g / kg) 125 ⁇ g / ml solution.
  • the LLC cells in the logarithmic growth phase were collected, centrifuged at 1000 r/min for 5 min, the supernatant was removed, and the cells were suspended in a physiological saline solution to adjust the cell concentration to 8.5 ⁇ 10 6 /ml.
  • the cell suspension was inoculated into the tail vein, and the number of cells inoculated was 1.7 ⁇ 10 6 cells/room, and the inoculation volume was 0.2 ml/head.
  • mice Ninety healthy and flexible C57BL/6 mice were randomly divided into 5 groups, 18 in each group, 9 out of each group were selected as the 30-day administration group, and the remaining 9 were used as the 60-day administration group. On the first day, the administration of the mice was started after the end of modeling, and the treatment was administered for 30 days and administered for 60 days, respectively, and the effect of the administration cycle on the therapeutic effect was examined.
  • the specific grouping and administration methods are shown in Table 1.
  • Tumor survival rate ILS (MSTT/MSTV-1) ⁇ 100%
  • the tumor inhibition rate of LLC lung metastasis mice was calculated by recording the number of lung metastasis nodules.
  • the effect of rLZ-8 on the tumor inhibition rate of LLC lung metastasis mice was investigated.
  • the specific experimental data are shown in Table 2.
  • rLZ-8 had a certain therapeutic effect on the lung metastasis model of mouse LLC lung cancer cells.
  • the rLZ-8 medium dose group had significant difference compared with the model group, which was statistically significant (p ⁇ 0.05).
  • Example 2 Effect of rLZ-8 on tumor morphology and tumor inhibition rate in nude mouse laryngeal carcinoma model and nude mouse glioma model
  • mice Male BALB/c-nude nude mice, 4-6 weeks old, weighing 18-22 g, were purchased from Beijing Huakang Biotechnology Co., Ltd., animal license number: SCXK Jing 2009-0004. Raised under SPF-grade aseptic conditions. During the experiment, the room temperature was controlled at (20 ⁇ 2) ° C, humidity 48%, and 12 hours alternately. The drinking water of the nude mice and the feed of the nude mice are sterile, and the experimental operations are completed in the ultra-clean platform to ensure the environment is sterile. Human laryngeal carcinoma cell line Hep-2 and rat glioma cell line 9L were all preserved by the Institute of Pharmacy. DMEM, fetal calf serum, PBS, trypsin, DMSO, 0.9% NaCl solution, pH 7.6 Tris-HCl buffer wash, 0.05% trypsin, rLZ-8.
  • DMEM fetal calf serum
  • PBS trypsin
  • DMSO 0.9% NaC
  • CO 2 constant temperature incubator inverted microscope, pipette, tweezers, ultra-clean workbench, low-speed centrifuge, ultra-low temperature freezer storage cabinet, electronic balance, fresh-keeping cabinet, refrigerator, sterilizer, water bath.
  • Flow cytometry disposable medical sterile gloves, medical sterile cotton, 50 ml centrifuge tube, pipette tip, cryotube, 10 cm cell culture plate, culture flask, 1.5 ml EP tube, cell counting plate.
  • Disposable 1ml syringe mouse litter, rat food.
  • Animal model establishment Hep-2 cells and 9L glioma cells in logarithmic growth phase were collected and centrifuged at 1000r/min for 5min respectively. The supernatant was removed and the cells were suspended in serum-free DMEM medium to adjust the cell concentration. of 2.5 ⁇ 10 7 / ml. The cell suspension was inoculated subcutaneously into the right flank of the mouse, and the number of cells inoculated was 5 ⁇ 10 6 /head, and the inoculation volume was 0.2 ml/head. A nude mouse model of subcutaneous transplantation was established, and the cells were administered in groups 24 hours later.
  • rLZ-8 anti-xenal laryngeal cancer in vivo study grouped into: healthy and flexible BALB/C-nude nude mice 80, weighing 18-22g, then divided into 4 groups, 20 in each group, each group randomly selected 10 Only, as the 60-day administration group, the remaining 10 were used as the 90-day administration group, and were divided into the model control group and the rLZ-8 low, medium and high dose treatment groups, wherein the administration methods were: model control tail vein Normal saline was injected once a day; rLZ-8 low, medium and high dose groups were injected with 125 ⁇ g/kg, 250 ⁇ g/kg, 500 ⁇ g/kg rLZ-8 solution once a day; the administration time was 60 days. 90 days.
  • rLZ-8 anti-malignant mouse glioma in vivo study grouped into: healthy and flexible BALB/C-nude nude mice 80, weighing 18-22g, then divided into 4 groups, each group of 20, each group randomly selected Ten rats were used as the 60-day administration group, and the remaining 10 were used as the 90-day administration group, which were divided into model group, rLZ-8 low, medium and high dose treatment groups; the administration methods were: model control group, tail vein injection Normal saline once a day; rLZ-8 low, medium and high dose groups were injected with 125 ⁇ g/kg, 250 ⁇ g/kg, 500 ⁇ g/kg rLZ-8 solution once a day; the days of administration were 60 days and 90 days. day.
  • rLZ-8 The effect of rLZ-8 on the volume of subcutaneous xenograft tumor of laryngeal carcinoma in nude mice: After inoculation of human laryngeal carcinoma cell Hep-2, the tumor volume of each group showed an increasing trend. The tumor volume was analyzed with time. Less than the negative control group, the middle and high dose groups of rLZ-8 had significant differences in the volume of subcutaneous xenograft tumors of laryngeal carcinoma compared with the model group.
  • rLZ-8 The effect of rLZ-8 on the weight of laryngeal carcinoma transplanted subcutaneously in nude mice: tumor cells were sacrificed 60 days after inoculation and 90 days after administration, tumor tissues were excised, weighed, and the difference in tumor weight was compared to calculate tumor suppression. Rate (see Table 3).
  • the experimental results showed that with the treatment time, the tumor weights of all the treatment groups except the model group showed a trend of less gradually, among which, the trend of rLZ-8 was the most obvious, and the tumor inhibition rate reached 91.12% after 60 days of administration. Compared with the model group, there was a significant difference (p ⁇ 0.05); the tumor inhibition rate reached 96.14% at 90 days of administration, and there were significant differences in the model group, which were statistically significant. At the same time, a mouse model in which the subcutaneous xenograft tumor disappeared appeared in the rLZ-8 medium dose group, which is unexpected in the present invention.
  • the effect of rLZ-8 on the volume of subcutaneous xenograft tumors in glioma nude mice After the inoculation of rat glioma 9L cells, the tumor volume of each group showed an increasing trend, and the change of tumor volume with time was analyzed. It can be seen that the tumor volume of each treatment group was smaller than that of the model control group, and the tumor volume of each group was significantly different from that of the model control group. The inhibition effect of the rLZ-8 medium dose group was the most significant.
  • rLZ-8 The effect of rLZ-8 on the weight of subcutaneous xenografts in glioma nude mice: 60 days after tumor cells were inoculated and 90 days after administration, the tumor tissues were excised, weighed, and the differences between tumor weights were calculated. The rate of inhibition of tumors (see Table 4).
  • the experimental results showed that the tumor weight of each group was smaller than that of the model control group, and there was a difference compared with the model control group. There was a significant difference in the tumor inhibition rate between the rLZ-8 middle dose group and the model group, and there was a statistically significant mouse model in which the subcutaneous xenograft tumor disappeared in the rLZ-8 middle dose group. Unexpectedly, it can be seen that rLZ-8 has more clinical therapeutic value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明公开了重组灵芝免疫调节蛋白rLZ-8在治疗肺癌、喉癌和脑胶质瘤中的应用。本发明分别通过尾静脉注射方法建立了肺癌小鼠模型、皮下移植瘤方法建立了喉癌及脑胶质瘤的裸鼠动物模型研究rLZ-8的体内抗肿瘤作用，治疗结果表明rLZ-8对肺癌、喉癌及脑胶质瘤具有抗肿瘤作用。

Description

重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用 技术领域
本发明属于生物医药领域，涉及重组灵芝免疫调节蛋白(rLZ-8)在制备治疗抗肿瘤药物中的应用，重点涉及重组灵芝免疫调节蛋白(rLZ-8)在抗肺癌、喉癌以及脑胶质瘤的生长的治疗作用，提高改善荷瘤小鼠生活状态及提高荷瘤小鼠存活率。
背景技术
肺癌是来源于支气管或细支气管表皮细胞的恶性肿瘤，占肺实质恶性肿瘤的90-95％。肺癌是肺部最常见的原发性恶性肿瘤。世界卫生组织调查报告，许多国家和地区，肺癌的发病率占恶性肿瘤的首位。本病多在40多岁以上发病，发病年龄高峰在60-79岁之间。男女患病率为2.3:1。肺癌起源于支气管粘膜上皮，局限于基底膜内者成为原位癌，可向支气管腔内或/和临近的肺组织生长，并可通过淋巴血行或经支气管转移扩散。肺癌主要有两大基本类型，即小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)。其中非小细胞肺癌(NSCLC)发生更多，约80％的肺癌患者属于这种类型。目前小细胞肺癌患者主要用化学疗法治疗，外科治疗对这种类型肺癌患者并不起主要作用。外科治疗主要适用于非小细胞肺 癌患者。
肺癌的主要原因是由于外界环境危险因素暴露的逐渐增加，如吸烟和被动吸烟人群的大范围存在、呼吸系统疾病(肺结核、慢性阻塞性肺疾患、哮喘等)的增加、城市大气污染的增加、厨房油烟的不完全处理、新鲜蔬菜和水果摄入量减少等。目前肺癌治疗方面尚未做到早期发现，早期诊断及早期治疗，多数病人在第一次外科诊断时病变已属晚期。所以，尽快开发和研究出肺癌的有效治疗药物是目前科学研究的一个重大而又艰巨的任务。
喉癌(Laryngocarcinoma)为消化系统癌症的一种。大多数的喉癌都是鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)，所占比例约95％左右，可见喉癌主要是于喉上皮的鳞状上皮细胞上形成。喉癌可以在喉的任何一个位置形成，而喉癌的康复率很受癌细胞位置的影响。喉癌主要可分为三个基本类型：声门型(声带)，声门上型(声襞)和声门下型。喉癌以声门型最为普遍，其次是声门上型，而最少出现的就是声门下型。据统计资料，喉癌占全身恶性肿瘤的1.2-1.6％，占头颈恶性肿瘤的3.3-8.1％。喉癌具有高发病率、高复发率的特点，男性发病居多，每年新发病人为3.5-5.5/10万，女性为0.6/10万，发达国家发病率较高，死亡率约为2.4/10万，女性为0.3/10万。呼吸道肿瘤中，喉癌发病率居第2位。
目前喉癌的治疗主要是通过手术、放疗、化疗、生物治疗手段的合理应用，以达到最大程度消灭肿瘤、保留喉功能、提高患者生存率。其中 手术治疗是喉癌的首选治疗方法，但是手术治疗不可避免地造成喉部损害，同时也会影响病人的生活质量。喉癌的放疗适用于病变＞1cm、表浅、无转移的声门上型喉癌，且全身情况差，不宜手术者。但是喉癌细胞对辐射的灵敏度不同，有些抗辐射的细胞即使在高强度的照射下也会存活。喉癌的诱导化疗对器官保留有重要作用，目前临床试验结果显示，化疗可以提高保喉率，减少远处转移，对患者总的生存率并无提高。
脑胶质瘤(Glioma)是胶质瘤的一种，胶质瘤是一种始于脑部或者脊柱的肿瘤，它被称为胶质瘤的原因是其由胶质细胞癌变产生，其中脑部的胶质瘤是最常见的。脑部肿瘤与中枢神经系统原发性和转移性的异质群体有关，是一种危及生命，存活率较低的疾病。原发性恶性脑瘤的年发病率大约是24000例。恶性神经胶质瘤是发生于神经胶质部位的肿瘤，发生率占脑瘤的70％。
目前脑胶质瘤的治疗方法主要包括手术治疗、放疗和化疗。脑胶质瘤的特点是呈弥漫性、侵袭性向周围脑组织浸润生长,与周围组织无明显界限，故手术不能完全切除肿瘤，还可能激活处于休眠期的瘤细胞，使之迅速浸入增殖期，导致切除后易复发，多于瘤床2cm范围内复发,治疗效果较差。放疗在治疗脑胶质瘤方面，作用十分有限，当放射剂量达到73-80Gy时，才能对胶质瘤细胞形成有效杀伤，而正常脑组织所能耐受剂量仅有60Gy，而且部分恶性脑瘤需进行大范围脑部放射线治疗或全颅及脊椎放射治疗，故放疗对病人学习和记忆能力均会造成一定的损伤。目前 由于脑胶质瘤对化疗药物产生一定的耐药性并且大部分治疗脑胶质瘤的化疗药物具有较强的毒副作用，故脑胶质瘤的化疗药物的疗效较低。
灵芝(Ganodermalucidum)是一种药用真菌，作为中药材已有数千年的药用历史，据报道，灵芝有免疫调节以及抗肿瘤作用。灵芝中的活性成分之一灵芝免疫调节蛋白(LZ-8)是从赤灵芝中提取的真菌免疫调节蛋白，本论文提供的重组灵芝免疫调节蛋白(Recombinant Ganodermalucidumimmunoregulatory protein，rLZ-8)是重新设计编码并全合成LZ-8基因，并通过毕赤酵母表达系统重组表达并纯化获得的蛋白样品，以往研究表明，rLZ-8也具有一定的抗肿瘤活性，例如，rLZ-8可有效杀伤人肺腺癌肿瘤细胞(A549)，人胃腺癌细胞(SGC7901)等肿瘤细胞株，但关于rLZ-8对肺癌、喉癌及脑胶质瘤的治疗作用尚未见报道。本发明提出rLZ-8对肺癌、喉癌及脑胶质瘤均有显著的治疗效果。
发明内容
本发明涉及rLZ-8在制备治疗肺癌、喉癌及脑胶质瘤药物中的应用，通过一系列的实验手段及结果表明rLZ-8对肺癌、喉癌及脑胶质瘤均具有显著的抑制作用，具体发明内容如下：
(1)采用健康灵活C57BL/6小鼠建立了肺癌转移瘤模型。设计了rLZ-8体内抗肺癌实验分组，其中包括正常对照组、模型组、rLZ-8低剂量组、rLZ-8中剂量组、rLZ-8高剂量组在内的5个实验组，各组用药剂量详见实施例1，以荷瘤小鼠生存时间作为考察对象，分别给药30天与 给药60天后，统计各组小鼠肺结节数和生存情况，计算rLZ-8对荷瘤小鼠肿瘤抑制率以及荷瘤小鼠存活率。
(2)采用健康灵活的BALB/c-nude裸鼠建立喉癌裸鼠皮下移植瘤模型。其中，rLZ-8体内抗喉癌研究设计了包括模型对照组(生理盐水)、rLZ-8低剂量组、rLZ-8中剂量组、rLZ-8高剂量组在内的4个实验组，其中rLZ-8低、中、高剂量组的剂量分别为125μg/kg、250μg/kg、500μg/kg，给药时间为60天与90天。治疗效果指标主要记录体重、瘤重、瘤体积并以瘤重为依据计算肿瘤抑制率等。分别将数据进行了统计学分析得出rLZ-8在治疗喉癌的效果显著。
(3)采用健康灵活的BALB/c-nude裸鼠建立脑胶质瘤裸鼠皮下移植瘤模型。设计了包括模型对照组(生理盐水)、rLZ-8低剂量组、rLZ-8中剂量组、rLZ-8高剂量组在内的4个实验组，其中，rLZ-8低、中、高剂量组的剂量分别为125μg/kg、250μg/kg、500μg/kg。给药天数为60天与90天。治疗效果指标主要记录体重、瘤重、瘤体积并以瘤重为依据计算肿瘤抑制率等。分别将数据进行了统计学分析得出rLZ-8在治疗脑胶质瘤的效果显著。
经过一系列rLZ-8对肺癌、喉癌及脑胶质瘤皮下移植瘤小鼠实验动物模型的药效学实验结果表明，rLZ-8能够改善荷瘤小鼠的生存状态，抗肿瘤效果显著。
本发明有益效果：本发明采用体内试验方式验证rLZ-8在治疗肺癌、 喉癌及脑胶质瘤的应用，通过一系列的药效学实验，结果表明，rLZ-8治疗肺癌、喉癌及脑胶质瘤效果显著，其中，rLZ-8在肺癌的治疗中不仅提高了肿瘤抑制率延长荷瘤小鼠的存活时间，而且出现了一定比例的肺癌结节数消失的荷瘤小鼠模型，这是发明人意想不到的；同时在治疗喉癌及脑胶质瘤的应用中同样出现了皮下实体瘤消失的效果。
本发明的这些目的，特点，和优点将会在下面的具体实施方式，附图，和权利要求中详细的揭露。
具体实施方式
实施例1rLZ-8对肺转移小鼠(LLC)肿瘤抑制率及其荷瘤小鼠存活率的影响
1药物配制
将rLZ-8蛋白原液的浓度为1mg/ml分别配置成rLZ-8低剂量(250μg/kg)31.25μg/ml溶液，rLZ-8中剂量(500μg/kg)62.5μg/ml溶液，为rLZ-8高剂量(1000μg/kg)125μg/ml溶液。
2肺癌小鼠模型的建立
收集对数生长期的LLC细胞，1000r/min离心5min，除去上清液，将细胞悬浮于生理盐水溶液中，调整细胞浓度为8.5×106/ml。取细胞悬液尾静脉接种于小鼠体内，接种细胞数为1.7×106个/只，接种体积为0.2ml/只。
3实验分组及给药方法
健康灵活的C57BL/6小鼠90只，随机分为5组，每组18只，每组选出9只作为30天给药组，剩余9只作为60天给药组。第1天小鼠造模结束后开始给药，分别给药治疗30天与给药60天，考察给药周期对治疗效果的影响。具体分组及给药方式见表1。
表1 实验分组及给药方法(包括给药30天与给药60天)
Figure PCTCN2015071893-appb-000001
4观察指标
4.1rLZ-8对LLC肺转移小鼠肿瘤抑制率与生存时间的影响
LLC小鼠肺癌细胞静脉接种于动物后，多数肿瘤细胞转移至肺部引起动物死亡，通过记录肺转移结节数，计算出LLC肺转移小鼠肿瘤抑制率，考察rLZ-8对LLC肺转移小鼠肿瘤抑制率的影响，以及通过比较动物生存时间来评价药物的治疗作用。
4.2rLZ-8对LLC肺转移小鼠中位生存期及其荷瘤存活率的影响
当累积生存率为0.5时所对应的生存时间，表示有且只有50％的个体可以活过这个时间。荷瘤存活率ILS＝(MSTT/MSTV-1)×100％
(MSTT和MSTV分别代表治疗组和对照组小鼠存活天数的中值 MST；MST＝1/2(X+Y)，X＝半数动物死亡天数，Y＝(半数+1)动物死亡天数)。
5实验结果与分析
rLZ-8对LLC细胞肺转移小鼠肿瘤抑制率与生存时间的影响。
给药60天后，通过记录肺转移结节数，计算出LLC肺转移小鼠肿瘤抑制率，考察rLZ-8对LLC肺转移小鼠肿瘤抑制率的影响，具体实验数据见表2。
表2 rLZ-8对LLC细胞肺转移小鼠肿瘤抑制率的影响(n＝9)
Figure PCTCN2015071893-appb-000002
注：*与模型组比较具有显著性差异p<0.05。
以上数据显示，rLZ-8对小鼠LLC肺癌细胞肺转移模型有一定的治疗效果，其中，rLZ-8中剂量组与模型组比较存在明显差异，具有统计学意义(p<0.05)。
实施例2：rLZ-8对裸鼠喉癌模型及裸鼠脑胶质瘤模型的肿瘤形态及抑瘤率影响
实验方法
(1)实验材料与试剂
选用4-6周龄的BALB/c-nude雄性裸鼠，体重18-22g，购自北京华阜康生物科技股份有限公司，动物许可证编号：SCXK京2009-0004。在SPF级无菌条件下饲养。实验期间控制室温在(20±2)℃，湿度48％，12小时交替照明。裸鼠饮用水以及其裸鼠饲料均为无菌，实验操作均在超净台内完成，保证环境处于无菌状态。人喉细胞癌细胞株Hep-2，大鼠脑胶质瘤细胞株9L，均由药学院实验室保存。DMEM、胎牛血清、PBS、胰酶、DMSO、0.9％NaCl溶液、pH7.6Tris-HCl缓冲液洗涤、0.05％胰蛋白酶、rLZ-8。
(2)仪器设备与器具
CO2恒温培养箱、倒置显微镜、移液器、镊子、超净工作台、低速离心机、超低温冷冻储存柜、电子天平、保鲜柜、冰箱、灭菌锅、水浴锅。流式细胞仪，一次性医用无菌手套、医用无菌棉花、50ml离心管、枪头、冻存管、10cm细胞培养板、培养瓶、1.5mlEP管、细胞计数板。一次性1ml注射器、小鼠垫料、鼠粮。
(3)实验分组及给药方式
动物模型建立：收集对数生长期的Hep-2细胞、9L脑胶质瘤细胞，分别1000r/min离心5min，除去上清液，将细胞悬浮于无血清的DMEM培养基溶液中，调整细胞浓度为2.5×107/ml。取细胞悬液接种于小鼠右侧肋部皮下，接种细胞数为5×106个/只，接种体积为0.2ml/只，建立裸 鼠皮下移植瘤模型，24小时后分组给药。
rLZ-8抗裸鼠喉癌体内研究分组给药方式为：健康灵活BALB/C-nude裸鼠80只，体重18-22g，随即分为4组，每组20只，每组随机选出10只，作为60天给药组，其余10只作为90天给药组，分为模型对照组、rLZ-8低、中、高剂量治疗组，其中，给药方式分别为：模型对照组尾静脉注射生理盐水，每天一次；rLZ-8低、中、高剂量组分别尾静脉注射剂量为125μg/kg、250μg/kg、500μg/kg的rLZ-8溶液，每天一次；给药时间为60天与90天。
rLZ-8抗裸鼠脑胶质瘤体内研究分组给药方式为：健康灵活BALB/C-nude裸鼠80只，体重18-22g，随即分为4组，每组20只，每组随机选出10只，作为60天给药组，其余10只作为90天给药组，分为模型组，rLZ-8低、中、高剂量治疗组；给药方式分别为：模型对照组尾静脉注射生理盐水，每天一次；rLZ-8低、中、高剂量组分别尾静脉注射剂量为125μg/kg、250μg/kg、500μg/kg的rLZ-8溶液，每天一次；给药天数为60天与90天。
实验期间观察小鼠的生活状态，每天称量体重，每周测量肿瘤体积2次。实验结束时剥离瘤体，把瘤体放到天平上称重并记录；按照公式肿瘤生长抑制率＝(生理盐水组瘤体重的平均值－给药组瘤体重的平均值)/生理盐水组瘤体重的平均值，计算rLZ-8对原位肿瘤生长的抑制率。
实验结果
rLZ-8对喉癌裸鼠皮下移植瘤瘤体积的影响：人喉癌细胞Hep-2接种后，各组肿瘤体积均呈增长趋势，分析其肿瘤体积随时间的变化可知，各组肿瘤体积均小于阴性对照组，其中rLZ-8中、高剂量组对喉癌皮下移植瘤瘤体积与模型组相比均有显著性差异。
rLZ-8对喉癌裸鼠皮下移植瘤瘤重的影响：肿瘤细胞接种后给药60天与给药90天后处死，将肿瘤组织剥离出来，称重，比较瘤重间的差异，计算肿瘤抑制率(见表3)。
表3 rLZ-8对喉癌模型裸鼠肿瘤重量的影响(n＝10)
Figure PCTCN2015071893-appb-000003
*与模型组比较具有显著性差异p<0.05；
实验结果表明，随着治疗时间的推移，除模型组外其余各治疗组肿瘤瘤重都出现逐渐较少的趋势，其中，rLZ-8的趋势最为明显，给药60天抑瘤率达到91.12％，分别与模型组比较具有显著性差异p<0.05；在给药90天抑瘤率达到了96.14％，分别于模型组比较均具有显著地差异，具有统计学意义。同时rLZ-8中剂量组出现了3只皮下移植瘤瘤体消失的小鼠模型，这是本发明意想不到的。
rLZ-8对脑胶质瘤裸鼠皮下移植瘤瘤体积的影响：大鼠脑胶质瘤9L细胞接种后，各组肿瘤体积均呈增长趋势，分析其肿瘤体积随时间的变化 可知，各治疗组肿瘤体积均小于模型对照组，且各组肿瘤体积与模型对照组相比均有极显著性差异，rLZ-8中剂量组的抑制作用最为显著。
rLZ-8对脑胶质瘤裸鼠皮下移植瘤瘤重的影响：肿瘤细胞接种后给药60天与给药90天后处死，将肿瘤组织剥离出来，称重，比较瘤重间的差异，计算对肿瘤的抑制率(见表4)。
表4 rLZ-8对脑胶质瘤模型裸鼠肿瘤重量的影响(n＝10)
Figure PCTCN2015071893-appb-000004
**与模型组相比p＜0.01。
实验结果表明：各组瘤重均小于模型对照组，与模型对照组相比存在差异。其中rLZ-8中剂量组与模型组的抑瘤率存在显著差异，具有统计学意义，同时rLZ-8中剂量组出现了4只皮下移植瘤瘤体消失的小鼠模型，这是本发明意想不到的，可以看出，rLZ-8更具有临床治疗价值。
通过上述实施例，本发明的目的已经被完全有效的达到了。熟悉该项技艺的人士应该明白本发明包括但不限于附图和上面具体实施方式中描述的内容。任何不偏离本发明的功能和结构原理的修改都将包括在权利要求书的范围中。

Claims (6)

  1. 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用。
  2. 如权利要求1所述肺癌，其特征在于肺癌包括小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)。
  3. 如权利要求1所述喉癌，其特征在于喉癌包括声门型(声带)，声门上型(声襞)和声门下型。
  4. 如权利要求1所述脑胶质瘤，其特征在于脑胶质瘤的发生部位包括脑部或者脊柱。
  5. 如权利要求1所述药物，其特征在于该药物制剂核心成分是由权利要求1所述的重组灵芝免疫调节蛋白(rLZ-8)和任选的药学可接受的辅剂组成。
  6. 如权利要求1、4所述的药物制剂给药途径为口服和非肠道给药，其中，口服包括口服液，片剂，丸剂和胶囊；非肠道给药包括外用药和注射剂。
PCT/CN2015/071893 2014-06-18 2015-01-30 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用 WO2015192663A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410270401.9A CN104043105A (zh) 2014-06-18 2014-06-18 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用
CN201410270401.9 2014-06-18

Publications (1)

Publication Number Publication Date
WO2015192663A1 true WO2015192663A1 (zh) 2015-12-23

Family

ID=51496827

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/071893 WO2015192663A1 (zh) 2014-06-18 2015-01-30 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用

Country Status (2)

Country Link
CN (2) CN104940898A (zh)
WO (1) WO2015192663A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352345A1 (en) * 2017-04-08 2019-11-21 Xitian Zhang Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104940898A (zh) * 2014-06-18 2015-09-30 张喜田 重组灵芝免疫调节蛋白rLZ-8在制备治疗脑胶质瘤药物中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104043105A (zh) * 2014-06-18 2014-09-17 张喜田 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101475632B (zh) * 2008-01-03 2012-01-04 张喜田 具有抗肿瘤作用的重组灵芝免疫调节蛋白及其药物制剂
CN103417953B (zh) * 2013-08-16 2015-06-17 张喜田 重组灵芝免疫调节蛋白(rLZ-8)在制备治疗黑色素瘤药物中的应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104043105A (zh) * 2014-06-18 2014-09-17 张喜田 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHIEN-TING WU ET AL.: "Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway", CARCINOGENESIS, vol. 32, no. 12, 7 October 2011 (2011-10-07), pages 1890 - 1896, XP055245155, ISSN: 0143-3334 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352345A1 (en) * 2017-04-08 2019-11-21 Xitian Zhang Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof
US12077566B2 (en) * 2017-04-08 2024-09-03 Xitian Zhang Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof

Also Published As

Publication number Publication date
CN104940898A (zh) 2015-09-30
CN104043105A (zh) 2014-09-17

Similar Documents

Publication Publication Date Title
TWI598104B (zh) Use of Antrodia cinnamomea extract to improve side effects of chemotherapy
CN109224071A (zh) 含有盐酸小檗碱和pd1-抗体的预防和/或治疗肿瘤的联用药物
WO2015192663A1 (zh) 重组灵芝免疫调节蛋白rLZ-8在治疗肺癌，喉癌和脑胶质瘤中的应用
CN107773753B (zh) 一种含有美洲大蠊和博来霉素的药物及其应用
US20130164382A1 (en) Ejaculum of animals as medicinal material and uses thereof in medicaments for treatment of diseases such as tumors, depression, etc
CN106974906A (zh) 穿心莲内酯在制备提高博来霉素抗肿瘤效果的药物中的应用
CA2894948C (en) Use of antisecretory factor (af) in glioblastoma treatment
CN114796183B (zh) 益母草碱在制备用于预防或治疗呼吸系统疾病的药物中的应用
CN114788822A (zh) 野黄芩素在制备治疗胰腺癌药物中的应用
Cao et al. A preliminary study on the anticancer efficacy of Caulis spatholobi compound 1802.
CN113908149A (zh) 刺芒柄花素在制备防治急性肺损伤药物中的用途
WO2022151335A2 (zh) 一种高压氧联合免疫药物用于肿瘤治疗的方法
WO2023029141A1 (zh) 奥替尼啶双盐酸盐在制备抗肿瘤药物中的应用
CN118236359A (zh) 硫酸特布他林在防治肠缺血再灌注损伤中的应用
CN110856718A (zh) 苯并异硒唑衍生物与铂类药物联合用于制备治疗肿瘤药物与术后肿瘤复发药物中的应用
CN113304249B (zh) 胸腺素β4在制备肺纤维化合并肺癌病治疗药物中的应用
CN113350333B (zh) 一种egcg联合用药及其医药用途
CN110652511B (zh) 中乌宁在制备防治肾功能衰竭药物中的应用
CN113244208B (zh) Hpa在制备治疗非酒精性脂肪性肝病药物中的应用
JP2017119677A (ja) 肺癌の治療に用いられる医薬組成物
RU2437692C2 (ru) Способ лечения солидных злокачественных новообразований и их метастазов
Yang et al. Neoadjuvant Arterial Infusion Chemotherapy Combined With Immunotherapy in Treating Locally Advanced Lower Esophageal/Esophagogastric Junction Cancer
WO2021159546A1 (zh) 一种预防和/或治疗神经母细胞瘤的多肽药物及其用途
CN104353061B (zh) 血小板生成素在辐射造成肠组织损伤中的应用
CN115708840A (zh) 神香草提取物在制备抗肺癌药物中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15809295

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15809295

Country of ref document: EP

Kind code of ref document: A1