WO2016095503A1 - 一株戈氏梭菌驯化株的应用 - Google Patents

一株戈氏梭菌驯化株的应用 Download PDF

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WO2016095503A1
WO2016095503A1 PCT/CN2015/083573 CN2015083573W WO2016095503A1 WO 2016095503 A1 WO2016095503 A1 WO 2016095503A1 CN 2015083573 W CN2015083573 W CN 2015083573W WO 2016095503 A1 WO2016095503 A1 WO 2016095503A1
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dcg
docetaxel
hncv
tumor
strain
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邢来田
王勇
魏明谦
邢艳秋
李莲莲
邢立超
邓广霞
孟红
黄涛
杨冬霞
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Shandong Chuangxin Pharmaceutical Research & Development Co Ltd
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Shandong Chuangxin Pharmaceutical Research & Development Co Ltd
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Priority to US15/525,046 priority Critical patent/US10314809B2/en
Priority to EP15869014.9A priority patent/EP3235501B1/en
Priority to AU2015366914A priority patent/AU2015366914B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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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  • the invention relates to the application of a variant of Clostridium ghonii (DCG), in particular to the preparation of a domesticated strain of Clostridium jejuni in the treatment of non-small cell lung carcinoma (NSCLC) drugs.
  • DCG Clostridium ghonii
  • NSCLC non-small cell lung carcinoma
  • the central area of solid tumors is characterized by increased interstitial pressure, decreased cell pH, low oxygen content, and tumor cell heterogeneity.
  • the tumor microenvironment is also an important cause of tumor metastasis, invasion and insensitivity to traditional radiotherapy and chemotherapy.
  • the area of hypoxia or necrosis contained in solid tumors is not rich in blood supply from the hypoxic area, and the high interstitial pressure in the center of the tumor makes it difficult for tumor cells in the hypoxic area to obtain effective drug concentration. A place that cannot be effectively treated.
  • radiotherapy relies on the formation of oxygen free radicals, causing DNA damage in mitotic cells, so the lack of oxygen free radicals in the tumor center reduces DNA damage during treatment.
  • most chemotherapeutic drugs are only effective in rapidly dividing tumor cells, but have poor efficacy (fibronectin and collagen) on hypoxic tumor cells, mesenchymal cells, and tumor tissue structures that have stopped dividing.
  • traditional chemoradiotherapy often lacks a specific blow to tumor cells.
  • DCG is an obligate anaerobic Gram-positive Bacillus, which has two growth states: spores and vegetatives. DCG spores are extremely weak and can be used as a formulation to reduce the body's irritation.
  • DCG spores in the blood can easily enter the tumor tissue, germination and reproduction in the hypoxic/necrotic area of the tumor, and secretion of various hydrolase enzymes such as lipase and protease can effectively and indiscriminately destroy the tumor tissue.
  • various cells tumor cells, non-cancer stromal cells, active division, stem cells or quiescent cells
  • tumor structures fibronectin and collagen
  • the destruction of the tumor microenvironment effectively inhibits or reverses the tumor growth and destroys the tumorigenic condition, and can even lead to the complete destruction of the entire tumor, which provides a possibility for deep treatment of NSCLC.
  • the inflammatory response induced by DCG secretion hydrolase in the tumor tissue is directly involved in the destruction of tumor cells by reactive oxygen species, proteases and other degrading enzymes. More importantly, the inflammatory response can stimulate the body to produce a strong cellular immune response, destroying the microenvironment of the remaining tumor cells.
  • Chinese patent document CN103374538A (Application No. 201210310374.4) discloses a non-toxic non-pathogenic derivative strain of Clostridium ghonii which inhibits the growth, reversal or destruction of one or more solid tumors. a solid tumor.
  • the invention also relates to a composition comprising the derivative bacterial strain for targeting the dissolution of a solid tumor.
  • the present invention is directed to the deficiencies of the prior art and provides a domesticated strain of Clostridium grosvenii MW-DCG-HNCv-18.
  • a domesticated strain of Clostridium grosvenii MW-DCG-HNCv-18 is used in the preparation of a medicament for treating non-small cell lung cancer.
  • the domesticated Clostridium grosvenii MW-DCG-HNCv-18 strain was white on a reinforced Clostridium-agar medium, round, with a diameter of about 1 to 2 mm, rough edges, slightly sticky, and uniform size.
  • the vegetative body is a short straight long cell which grows alone, and the cell body is about 0.3-1.5 ⁇ m wide and 8-16 ⁇ m long, and Gram stain is positive.
  • the spore is located at the proximal end of the cell and is oval in shape.
  • This strain is a known strain and can be obtained from the Australian National Institute of Metrology with the strain accession number V12/001485.
  • a medicament for treating non-small cell lung cancer comprising a pharmaceutical composition prepared by acclimating a strain of Clostridium gamma MW-DCG-HNCv-18 and docetaxel as a medicinal ingredient with a pharmaceutically acceptable adjuvant.
  • the ratio of spores to docetaxel of the domesticated Clostridium grosvenii MW-DCG-HNCv-18 strain is 5 ⁇ 10 6 to 3.0 ⁇ 10 7 strains per mg of docetaxel. Spores.
  • domesticated Clostridium ghonii MW-DCG-HNCv-18 strain of Bacillus said ratio docetaxel per ml docetaxel combined Keduo Xi 1.0 ⁇ 10 7 th ⁇ 2.0 ⁇ 10 7 spore isolates.
  • the domesticated MW-DCG-HNCv-18 strain of Bacillus and Clostridium Ge ratio of docetaxel per ml docetaxel combined Keduo Xi 1.5 ⁇ 10 7 spore isolates.
  • the pharmaceutical dosage form for treating non-small cell lung cancer is a lyophilized powder injection.
  • the spore freeze-dried powder can be prepared by a low-temperature freeze-drying technique.
  • the spore lyophilized powder injection is used in combination with a solvent selected from the group consisting of Ringer's solution, physiological saline having a mass concentration of 0.9%, and a phosphate buffer solution.
  • a solvent selected from the group consisting of Ringer's solution, physiological saline having a mass concentration of 0.9%, and a phosphate buffer solution.
  • the solvent needs to meet non-toxic, injectable requirements. It is administered by intravenous, intraspinal, intramuscular, subcutaneous, intratumoral injection, and intraperitoneal administration.
  • the present invention finds for the first time that MW-DCG-HNCv-18 strain has a specific inhibitory effect on non-small cell lung cancer, and the inhibitory effect on non-small cell lung cancer is significantly better than other similar strains known in the prior art, and it is found through screening. When combined with docetaxel injection, the inhibitory effect on non-small cell lung cancer is more prominent, providing a new way to treat non-small cell lung cancer;
  • the DCG spores of the present invention have the characteristics of obligate anaerobic and can move to the hypoxic zone of the tumor, causing most of the oncolytic necrosis of the tumor.
  • the proliferation and movement of the bacteria are stagnant, and the edge Residual tumor tissue still has the potential for recurrence, while docetaxel can rapidly rupture tumor cells in the oxygen-rich environment at the margin of tumor tissue at lower doses.
  • chemotherapeutic drugs can destroy the tumor blood vessels, aggravate the degree of hypoxia inside the tumor, and is more conducive to DCG colonization and improve the oncolytic effect.
  • Figure 1 is a photograph of the anti-tumor effect of Example 1 MW-DCG-HNCv-18;
  • Example 2 is a tumor growth curve of each experimental group obtained by the experimental test of Example 2;
  • Fig. 3 is a photograph showing the results of the antitumor effect on the 20th day of the detection of Example 2.
  • the MW-DCG-HNCv-18 strain was obtained from the Australian National Institute of Metrology and the strain accession number was V12/001485.
  • the MW-DCG-LCv-26 strain was obtained from the Australian National Institute of Metrology, strain accession number V12/001486.
  • the MW-DCG-CCv-17 strain was obtained from the Australian National Institute of Metrology, strain accession number V12/001487.
  • Clostridium novyi–NT strain is derived from ATCC, strain accession number 7659 TM .
  • Cetuximab (5 mg/ml) was purchased from MERCK Serono, Germany, batch number: 132393, date of manufacture: February 2013.
  • Docetaxel was purchased from Qilu Pharmaceutical Co., Ltd., batch number: 3030062TA, production date: March 18, 2013.
  • Doxorubicin was purchased from Shenzhen Wanle Pharmaceutical Co., Ltd., batch number: 1206E4. Production date: April 2013.
  • Non-small cell lung cancer A549 cells were slowly injected into the female Balb/c-nu/nu nude mice (5 weeks old, 20 g) by subcutaneous injection of cells, and the right lower thigh was subcutaneously subcutaneously. When the tumor volume was as long as 300 mm 3 , the cells were in vivo. Anti-tumor test.
  • n 15, spores were injected into the tail vein on the first day, and the dose was 3 ⁇ 10 8 /(kg.body weight).
  • the long diameter (a) and transverse diameter (b) of nude mice xenografts were measured with vernier calipers, 3 to 4 times per week.
  • the general conditions of the tumor-bearing mice including weight, feeding, drinking water, mental state, behavior, fur color, excretion and death, were observed and recorded.
  • the tumor volume (V 1/2 ⁇ a ⁇ b 2 ) was calculated from the measured tumor long diameter and transverse diameter data for each measurement, and a curve was drawn to dynamically observe the tumor change.
  • Tumor inhibition rate (%) (control group average tumor weight - experimental group average tumor weight) / control group average tumor weight ⁇ 100%.
  • Tumor necrosis rate (%) cross-sectional area of the necrotic area / total tumor cross-sectional area x 100%.
  • Tumor oncolytic rate (%) number of tumor oncolytic animals / total number of animals in the group ⁇ 100%.
  • MW-DCG-HNCv-18 strain prolonged the average survival days of tumor-bearing mice, and Significantly longer than the average survival days of the C.novyi–NT group tumor-bearing mice.
  • the MW-DCG-HNCv-18 strain showed good efficacy and safety compared to the MW-DCG-LCv-26, MW-DCG-CCv-17 and C.novyi-NT strains.
  • Non-small cell lung cancer A549 cells were slowly injected into the female Balb/c-nu/nu nude mice (5 weeks old, 20 g) by subcutaneous injection of cells, and the right lower thigh was subcutaneously subcutaneously. When the tumor volume was as long as 300 mm 3 , the cells were in vivo. test.
  • the long diameter (a) and transverse diameter (b) of nude mice xenografts were measured with vernier calipers, 3 to 4 times per week, and the tumor volume of each group was calculated to observe the antitumor effect of the above bacteria (Fig. 2). At the same time, the weight, feeding, drinking water, mental state, behavior, fur color, excretion and death of the tumor-bearing mice were observed and recorded.
  • the tumor volume (V 1/2 ⁇ a ⁇ b 2 ) was calculated from the measured tumor long diameter and transverse diameter data for each measurement, and a curve was drawn to dynamically observe the tumor change.
  • Tumor inhibition rate (%) (control group average tumor weight - experimental group average tumor weight) / control group average tumor weight ⁇ 100%.
  • Tumor necrosis rate (%) area of necrotic area / whole tumor area ⁇ 100%.
  • Cure rate (%) number of animals completely eliminated by the tumor / total number of animals in the group ⁇ 100%.
  • the inhibition rate and necrosis rate of MW-DCG-HNCv-18 combined with docetaxel in non-small cell lung cancer-bearing mice model were significantly higher than those in other co-administration groups.
  • the tumor volume of MW-DCG-HNCv-18 combined with docetaxel-bearing mice showed inhibition on the sixth day of administration and remained at around 300 mm 3 , while other groups showed different degrees of growth (Fig. 2 ).
  • the tumor of MW-DCG-HNCv-18 combined with the docetaxel group was almost completely eliminated in 26.7% of tumor-bearing mice (Fig. 3), which was regarded as a cure, which was significantly higher than the other three co-administered groups.
  • MW-DCG-HNCv-18 combined with docetaxel can prolong the survival of tumor-bearing mice to 71.0 days, which is 2.9 times longer than that of the control mice, and significantly longer than MW-DCG-HNCv-18 combined with Cetuximab , MW-DCG-HNCv-18 combined with doxorubicin group and C.novyi-NT combined with docetaxel group. It can be seen that the combination of MW-DCG-HNCv-18 and docetaxel in the treatment of non-small cell lung cancer shows super-superposition.
  • Non-small cell lung cancer A549 cells were slowly injected into the female Balb/c-nu/nu nude mice (5 weeks old, 20 g) by subcutaneous injection of cells, and the right lower thigh was subcutaneously subcutaneously. When the tumor volume was as long as 300 mm 3 , the cells were in vivo. test.
  • docetaxel was injected into the tail vein at a dose of 20 mg/(kg. body weight).
  • docetaxel was injected into the tail vein at a dose of 20 mg/(kg. body weight).
  • n 15, the first day of tail vein injection of MW-DCG-HNCv-18 spore, the dose was 3.0 ⁇ 10 8 /(kg.body weight On the 4th and 11th day, docetaxel was injected into the tail vein at a dose of 20 mg/(kg. body weight).
  • docetaxel was injected into the tail vein at a dose of 20 mg/(kg. body weight).
  • docetaxel was injected into the tail vein at a dose of 20 mg/(kg. body weight).
  • n 15, the first day of tail vein injection of MW-DCG-HNCv-18 spore, the dose was 7.0 ⁇ 10 8 /(kg.body weight On the 4th and 11th day, docetaxel was injected into the tail vein at a dose of 20 mg/(kg. body weight).
  • the long diameter (a) and transverse diameter (b) of nude mice xenografts were measured with vernier calipers, 3 to 4 times per week. At the same time, the weight, feeding, drinking water, mental state, behavior, fur color, excretion and death of the tumor-bearing mice were observed and recorded.
  • the tumor volume (V 1/2 ⁇ a ⁇ b 2 ) was calculated from the measured tumor long diameter and transverse diameter data for each measurement, and a curve was drawn to dynamically observe the tumor change.
  • Tumor inhibition rate (%) (control group average tumor weight - experimental group average tumor weight) / control group average tumor weight ⁇ 100%.
  • Tumor necrosis rate (%) cross-sectional area of the necrotic area / total tumor cross-sectional area x 100%.
  • Cure rate (%) number of animals completely eliminated by the tumor / total number of animals in the group ⁇ 100%.
  • MW-DCG-HNCv-18 spore combined with docetaxel-2 MW-DCG-HNCv-18 spore combined with docetaxel-3 and MW-DCG-HNCv-18 spore combined with docetaxel-4
  • MW-DCG-HNCv-18 spore combined with docetaxel-4 group was used for anti-tumor. The role is best.
  • MW-DCG-HNCv-18 spores should be treated with 1.0 ⁇ 10 8 to 6.0 ⁇ 10 8 /(kg.body weight) for docetaxel.
  • a preferred range for non-small cell lung cancer wherein 3.0 x 10 8 / (kg. body weight) MW-DCG-HNCv-18 spores are the optimal dose for the treatment of non-small cell lung cancer in combination with docetaxel.

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Abstract

提供一株戈氏梭菌MW-DCG-HNCv-18在制备治疗非小细胞肺癌药物中的应用。还提供一种以戈氏梭菌MW-DCG-HNCv-18和多西他赛为活性成分的治疗非小细胞肺癌的药物。

Description

一株戈氏梭菌驯化株的应用 技术领域
本发明涉及一株戈氏梭菌驯化株(Derivatives of Clostridium ghonii,DCG)的应用，特别涉及戈氏梭菌驯化株在制备治疗非小细胞肺癌(Non-small cell lung carcinoma，NSCLC)药物中的应用，属于生物医药技术领域。
背景技术
目前实体肿瘤病理生理的理解越来越多的转向了其独特生长的微环境。实体瘤中心区以组织间压力升高、细胞pH降低、氧含量低以及肿瘤细胞异质性等为显著特点。肿瘤微环境也是肿瘤转移、侵润以及对对传统放化疗不敏感的重要原因。首先，实体肿瘤所包含的乏氧或坏死区域，由于乏氧区远离血管供血不丰富，并且肿瘤中心高组织间压的存在，使得乏氧区肿瘤细胞很难获得有效药物浓度，是传统化疗所不能有效治疗的地方。其次，放疗依赖于氧自由基的形成，造成有丝分裂细胞DNA的损伤，因此肿瘤中心氧自由基的缺乏减少了治疗过程中DNA损伤。第三，大多数化疗药物只对快速分裂肿瘤细胞有效，而对已停止分裂的乏氧肿瘤细胞、间质细胞及肿瘤组织结构等疗效差(纤连蛋白和胶原蛋白)。并且，传统放化疗常常缺乏对肿瘤细胞的特异性打击。
肿瘤厌氧微环境限制了传统放化疗及病毒疗法的疗效，但却为厌氧菌在肿瘤乏氧区内生长和发挥溶瘤特性提供了良好机会。DCG为专性厌氧革兰氏阳性芽孢杆菌，其存在两种生长状态:芽孢和营养体。DCG芽孢抗原性极弱，可作为制剂静脉注射降低对机体刺激。由于肿瘤血管组织结构紊乱，血液中的DCG芽孢极易进入肿瘤组织，在肿瘤低氧/坏死区萌发、繁殖，并分泌脂肪酶、蛋白酶等多种水解酶有效而不加区分的破坏肿瘤组织中各种细胞(肿瘤细胞、非癌间质细胞、活跃分裂、干细胞或静止细胞等)及肿瘤结构(纤连蛋白和胶原蛋白)，引起肿瘤大部分溶瘤坏死，实现肿瘤实质和间质的全部溶解消化。从根本上破坏并改变肿瘤微环境，这种肿瘤微环境的破坏有效抑制或逆转肿瘤生长和破坏再成瘤条件，甚至可导致整个肿瘤几乎完全破坏消失，为NSCLC深度治疗提供了可能。DCG分泌水解酶溶解瘤组织引发的炎症反应直接参与活性氧簇、蛋白酶及其他降解酶对肿瘤细胞的破坏作用。更重要的是，炎症反应可刺激机体产生强大细胞免疫反应，破坏残存肿瘤细胞微环境。
中国专利文献CN103374538A(申请号201210310374.4)公开了无毒非致病性戈氏梭菌(Clostridium ghonii)的衍生细菌菌株，其能抑制一种或多种实体瘤的生长、逆转或破坏一种或多种实体瘤。本发明还涉及包含所述衍生细菌菌株的组合物，用于靶向实体瘤的溶解。
但由于实体瘤的形成原因不同，因此不同菌株对实体瘤的作用都是特异性的。因此，进一步研究各种菌株对不同实体瘤的特异作用，成为本领域的研究热点。
发明内容
本发明针对现有技术的不足，提供一株驯化戈氏梭菌MW-DCG-HNCv-18菌株的应用。
一株驯化戈氏梭菌MW-DCG-HNCv-18菌株在制备治疗非小细胞肺癌药物中的应用。
驯化戈氏梭菌MW-DCG-HNCv-18菌株在强化梭菌-琼脂培养基上其菌落为白色，圆形，直径约为1～2mm，边缘粗糙，稍粘，其大小一致。其营养体为单独生长的短杆长直细胞，菌体宽约0.3～1.5μm、长8～16μm，革兰氏染色呈阳性。芽孢位于菌体近末端，呈卵圆形。该菌株为已知菌株，可以由澳大利亚国家计量研究院获得，菌株保藏号为V12/001485。
一种治疗非小细胞肺癌的药物，包括以驯化戈氏梭菌MW-DCG-HNCv-18菌株和多西他赛为药效成分，与药学上可接受的辅料制成的药物组合物。
根据本发明优选的，所述的驯化戈氏梭菌MW-DCG-HNCv-18菌株芽孢与多西他赛的比例为每毫克多西他赛联合5×106个～3.0×107个菌株芽孢。
进一步优选的，所述的驯化戈氏梭菌MW-DCG-HNCv-18菌株芽孢与多西他赛的比例为每毫克多西他赛联合1.0×107个～2.0×107个菌株芽孢。
更优选的，所述驯化戈氏梭菌MW-DCG-HNCv-18菌株芽孢与多西他赛的比例为每毫克多西他赛联合1.5×107个菌株芽孢。
根据本发明优选的，所述的治疗非小细胞肺癌的药物剂型为芽孢冻干粉针剂。芽孢冻干粉针可采用低温冷冻干燥技术制备。
根据本发明进一步优选的，所述芽孢冻干粉针剂与溶媒混合使用，溶媒选自林格氏液、质量浓度为0.9％的生理盐水、磷酸盐缓冲液。溶媒需要满足非毒性、可注射用的要求。通过静脉、脊柱内、肌注、皮下、肿瘤瘤内注射和腹腔等方式给药。
有益效果
1、本发明首次发现MW-DCG-HNCv-18菌株菌对非小细胞肺癌具有特异性抑制作用，对非小细胞肺癌抑制效果显著优于现有已知的其他类似菌株，并通过筛选发现其与多西他赛注射液联用时，对非小细胞肺癌抑制效果更加突出，为治疗非小细胞肺癌提供了新的途径；
2、本发明所述DCG芽孢具有专性厌氧的特点，并能向肿瘤乏氧区移动，引起肿瘤大部分溶瘤坏死，当它们到达富氧肿瘤边缘时，细菌增殖和移动发生停滞，边缘残存肿瘤组织仍有复发的潜能，而多西他赛可在较低剂量下杀伤肿瘤组织边缘富氧环境中快速分裂肿瘤细胞。同时，化疗药物又可通过破坏肿瘤血管，加剧肿瘤内部缺氧程度，更有利于DCG定殖，提高溶瘤效果。二者协同形成具有肿瘤靶向性的立体、高效靶向性的溶瘤效应，达到优势互补、内外夹击、产生“超叠加作用”。
附图说明
图1是实施例1MW-DCG-HNCv-18抗肿瘤效果照片；
图2是实施例2实验检测获得的各实验组的肿瘤生长曲线；
图3是实施例2检测第20天抗肿瘤效果的结果照片。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述，但本发明所保护范围不限于此。
菌株来源
MW-DCG-HNCv-18菌株来源于澳大利亚国家计量研究院，菌株保藏号为V12/001485。
MW-DCG-LCv-26菌株来源于澳大利亚国家计量研究院，菌株保藏号V12/001486。
MW-DCG-CCv-17菌株来源于澳大利亚国家计量研究院，菌株保藏号V12/001487。
Clostridium novyi–NT菌株来源于ATCC，菌株保藏号
Figure PCTCN2015083573-appb-000001
7659TM。
西妥昔单抗(5mg/ml)购自德国MERCK Serono，生产批号：132393，生产日期：2013年2月。
多西他赛购自齐鲁制药有限公司，批号：3030062TA，生产日期：2013年03月18日。
阿霉素购自深圳万乐药业有限公司，批号：1206E4。生产日期：2013年4月。
实施例1
研究方法：
①非小细胞肺癌模型建立
采用细胞皮下注射法将非小细胞肺癌A549细胞缓慢注入雌性Balb/c-nu/nu裸小鼠(5周龄，20g)右背部股外侧皮下处，待肿瘤体积长至300mm3时，进行体内抗肿瘤试验。
②动物分组与处理
对照组：n＝15只(n代表每组的动物数量)，尾静脉注射PBS缓冲液0.2ml；
MW-DCG-HNCv-18芽孢组：n＝15只，第1天尾静脉注射芽孢，剂量为3×108个/(kg.body weight)。
MW-DCG-LCv-26芽孢组：n＝15只，第1天尾静脉注射芽孢，剂量为3×108个/(kg.body weight)。
MW-DCG-CCv-17芽孢组：n＝15只，第1天尾静脉注射芽孢，剂量为3×108个/(kg.body weight)。
C.novyi–NT芽孢组：n＝15只，第1天尾静脉注射芽孢，剂量为3×108个/(kg.body weight)。
③检测指标
以游标卡尺测量裸鼠移植瘤长径(a)和横径(b)，每周3～4次。同时对荷瘤鼠一般情况包括体重、取食、饮水、精神状态、行为、皮毛颜色、排泄及死亡情况等进行观察记录。
④评价标准
根据每次测量的肿瘤长径和横径数据计算肿瘤体积(V＝1/2×a×b2)，绘制曲线，动态观察肿瘤的变化。
抑瘤率(％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％。
肿瘤坏死率(％)＝坏死区横截面面积/整个肿瘤横截面面积×100％。
肿瘤溶瘤率(％)＝发生肿瘤溶瘤动物数量/该组动物总数量×100％。
荷瘤鼠总体生存期：各组裸鼠经治疗后待其自然死亡，计算存活时间。
结果
Figure PCTCN2015083573-appb-000002
由此可见，(1)有效性：与MW-DCG-LCv-26、MW-DCG-CCv-17菌株相比，MW-DCG-HNCv-18对NSCLC荷瘤鼠模型肿瘤抑瘤率、坏死率和溶瘤率分别为51.1％、76.55％和100％，均显著高于MW-DCG-LCv-26、MW-DCG-CCv-17菌株。与C.novyi–NT菌株相比，MW-DCG-HNCv-18菌株的抑瘤率、坏死率和溶瘤率虽高于前者，但差异不显著。同时，研究结果显示，MW-DCG-HNCv-18芽孢组注射芽孢第35天后出现大面积坏死，而对照组肿瘤体积持续增大(图1)。
(2)安全性：与MW-DCG-LCv-26、MW-DCG-CCv-17和C.novyi–NT菌株相比，MW-DCG-HNCv-18菌株延长了荷瘤鼠平均存活天数，并显著长于C.novyi–NT组荷瘤鼠的平均存活天数。研究发现，C.novyi–NT组荷瘤鼠体重下降，而其它三组荷瘤鼠体重未见变化。
可见，与MW-DCG-LCv-26、MW-DCG-CCv-17和C.novyi–NT菌株相比，MW-DCG-HNCv-18菌株表现出良好的有效性和安全性。
实施例2
研究方法：
①非小细胞肺癌模型建立
采用细胞皮下注射法将非小细胞肺癌A549细胞缓慢注入雌性Balb/c-nu/nu裸小鼠(5周龄，20g)右背部股外侧皮下处，待肿瘤体积长至300mm3时，进行体内试验。
②动物分组与处理
对照组：n＝15只，尾静脉注射PBS缓冲液0.2ml；
多西他赛组：n＝15只，第4天和第11天尾静脉注射多西他赛，剂量为20mg/(kg.body weight)，多西他赛施用剂量为其最佳施用量。
MW-DCG-HNCv-18芽孢组：n＝15只，尾静脉注射MW-DCG-HNCv-18芽孢，3×108个/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合Cetuximab(西妥昔单抗)组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为3×108个/(kg.body weight)；第4天和第11天尾静脉注射Cetuximab(西妥昔单抗)，剂量均为70mg/(kg.body weight)，西妥昔单抗施用剂量为其最佳施用量。
MW-DCG-HNCv-18芽孢联合阿霉素组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为3×108个/(kg.body weight)；第4天和第11天尾静脉注射阿霉素，剂量均为10mg/(kg.body weight)，阿霉素施用剂量为其最佳施用量。
MW-DCG-HNCv-18芽孢联合多西他赛组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为3×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
C.novyi–NT芽孢联合多西他赛组：n＝15只，第1天尾静脉注射C.novyi–NT芽孢，剂量为3×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
③检测指标
以游标卡尺测量裸鼠移植瘤长径(a)和横径(b)，每周3～4次，并计算各组肿瘤体积，动态观察上述细菌的抗肿瘤效果(图2)。同时对荷瘤鼠体重、取食、饮水、精神状态、行为、皮毛颜色、排泄及死亡情况等进行观察记录。
④评价标准
根据每次测量的肿瘤长径和横径数据计算肿瘤体积(V＝1/2×a×b2)，绘制曲线，动态观察肿瘤的变化。
抑瘤率(％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％。
肿瘤坏死率(％)＝坏死区面积/整个肿瘤面积×100％。
治愈率(％)＝肿瘤完全消除动物数量/该组动物总数量×100％。
荷瘤鼠总体生存期：各组裸鼠经治疗后待其自然死亡，计算存活时间。
⑤结果
Figure PCTCN2015083573-appb-000003
由此可见，MW-DCG-HNCv-18联合多西他赛对非小细胞肺癌荷瘤鼠模型抑瘤率、坏死率，显著高于其他联合给药组。MW-DCG-HNCv-18联合多西他赛组荷瘤鼠肿瘤体积在给药第六天表现出抑制作用，并始终保持300mm3左右，而其它几组却表现出不同程度的增长(图2)。同时，MW-DCG-HNCv-18联合多西他赛组26.7％荷瘤鼠肿瘤几乎完全的消除(图3)，视为治愈，显著高于其他三个联合给药组。MW-DCG-HNCv-18联合多西他赛可延长荷瘤小鼠的存活期至71.0天，为对照组荷瘤鼠生存期的2.9倍，并显著长于MW-DCG-HNCv-18联合Cetuximab组、MW-DCG-HNCv-18联合阿霉素组和C.novyi–NT联合多 西他赛组。可见，MW-DCG-HNCv-18与多西他赛联合治疗非小细胞肺癌显示出超叠加作用。
实施例3
研究方法：
①非小细胞肺癌模型建立
采用细胞皮下注射法将非小细胞肺癌A549细胞缓慢注入雌性Balb/c-nu/nu裸小鼠(5周龄，20g)右背部股外侧皮下处，待肿瘤体积长至300mm3时，进行体内试验。
②动物分组与处理
对照组：n＝15只，尾静脉注射PBS缓冲液0.2ml；
MW-DCG-HNCv-18芽孢联合多西他赛-1组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢芽孢，剂量为0.5×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合多西他赛-2组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为1.0×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合多西他赛-3组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为2.0×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合多西他赛-4组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为3.0×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合多西他赛-5组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为5.0×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合多西他赛-6组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为6.0×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
MW-DCG-HNCv-18芽孢联合多西他赛-7组：n＝15只，第1天尾静脉注射MW-DCG-HNCv-18芽孢，剂量为7.0×108个/(kg.body weight)；第4天和第11天尾静脉注射多西他赛，剂量均为20mg/(kg.body weight)。
③检测指标
以游标卡尺测量裸鼠移植瘤长径(a)和横径(b)，每周3～4次。同时对荷瘤鼠体重、取食、饮水、精神状态、行为、皮毛颜色、排泄及死亡情况等进行观察记录。
④评价标准
根据每次测量的肿瘤长径和横径数据计算肿瘤体积(V＝1/2×a×b2)，绘制曲线，动态观察肿瘤的变化。
抑瘤率(％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％。
肿瘤坏死率(％)＝坏死区横截面面积/整个肿瘤横截面面积×100％。
治愈率(％)＝肿瘤完全消除动物数量/该组动物总数量×100％。
荷瘤鼠总体生存期：各组裸鼠经治疗后待其自然死亡，计算存活时间。
⑤结果
Figure PCTCN2015083573-appb-000004
由上述结果可见，MW-DCG-HNCv-18芽孢联合多西他赛-1组治疗非小细胞肺癌的抑瘤率、坏死率、治愈率和平均存活天数均显著低于其它MW-DCG-HNCv-18芽孢联合多西他赛组。MW-DCG-HNCv-18芽孢联合多西他赛-2组、MW-DCG-HNCv-18芽孢联合多西他赛-3组和MW-DCG-HNCv-18芽孢联合多西他赛-4组随着MW-DCG-HNCv-18芽孢剂量的增加其抑瘤率、坏死率、治愈率和平均存活天数逐渐增高，并以MW-DCG-HNCv-18芽孢联合多西他赛-4组抗肿瘤作用为最佳。与MW-DCG-HNCv-18芽孢联合多西他赛-4组相比，MW-DCG-HNCv-18芽孢联合多西他赛-7组抑瘤率和坏死率虽无显著差异，但其治愈率和平均存活天数均显著低于MW-DCG-HNCv-18芽孢联合多西他赛-4组。考虑MW-DCG-HNCv-18芽孢生产成本，研究认为应以1.0×108～6.0×108个/(kg.body weight)的MW-DCG-HNCv-18芽孢为其联合多西他赛治疗非小细胞肺癌的优选范围，其中以3.0×108个/(kg.body weight)MW-DCG-HNCv-18芽孢为联合多西他赛治疗非小细胞肺癌的最优剂量。

Claims (7)

  1. 一株驯化戈氏梭菌MW-DCG-HNCv-18菌株在制备治疗非小细胞肺癌药物中的应用。
  2. 一种治疗非小细胞肺癌的药物，其特征在于，包括以驯化戈氏梭菌MW-DCG-HNCv-18菌株和多西他赛为药效成分，与药学上可接受的辅料制成的药物组合物。
  3. 如权利要求2所述的药物，其特征在于，所述的驯化戈氏梭菌MW-DCG-HNCv-18菌株芽孢与多西他赛的比例为每毫克多西他赛联合5×106个～3.0×107个菌株芽孢。
  4. 如权利要求3所述的药物，其特征在于，所述的驯化戈氏梭菌MW-DCG-HNCv-18菌株芽孢与多西他赛的比例为每毫克多西他赛联合1.0×107个～2.0×107个菌株芽孢。
  5. 如权利要求4所述的药物，其特征在于，所述驯化戈氏梭菌MW-DCG-HNCv-18菌株芽孢与多西他赛的比例为每毫克多西他赛联合1.5×107个菌株芽孢。
  6. 如权利要求2所述的药物，其特征在于，所述的治疗非小细胞肺癌的药物剂型为芽孢冻干粉针剂。
  7. 如权利要求6所述的药物，其特征在于，所述芽孢冻干粉针剂与溶媒混合使用，溶媒选自林格氏液、质量浓度为0.9％的生理盐水、磷酸盐缓冲液。
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