WO2021189444A1 - 利福霉素类抗生素在制备抗黄热病毒感染药物中的应用 - Google Patents

利福霉素类抗生素在制备抗黄热病毒感染药物中的应用 Download PDF

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WO2021189444A1
WO2021189444A1 PCT/CN2020/081772 CN2020081772W WO2021189444A1 WO 2021189444 A1 WO2021189444 A1 WO 2021189444A1 CN 2020081772 W CN2020081772 W CN 2020081772W WO 2021189444 A1 WO2021189444 A1 WO 2021189444A1
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rifamycin
dosage form
yellow fever
fever virus
antibiotics
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PCT/CN2020/081772
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French (fr)
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赵平
钱汐晶
彭浩然
唐海琳
戚中田
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中国人民解放军海军军医大学
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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 belongs to the technical field of medicine, and specifically relates to the application of rifamycin antibiotics in the preparation of anti-yellow fever virus infection drugs.
  • Rifamycin antibiotics refer to semi-synthetic or synthetic antibiotics containing rifamycin derivatives, including a variety of antibiotics in the rifamycin family. They have broad-spectrum antibacterial effects against Mycobacterium tuberculosis, leprosy, streptococcus, and pneumonia Gram-positive bacteria such as cocci have a strong inhibitory effect, and it can also effectively inhibit certain gram-negative bacteria.
  • the natural rifamycin antibiotics were originally isolated from the culture broth of Streptomyces mediterraneanus by an Italian pharmaceutical company. Rifamycin B has strong activity and stable properties, but its clinical efficacy is not ideal. Later, by artificially changing the chemical structure of natural rifamycin B, a series of more potent antibiotics, called semi-synthetic rifamycins, were obtained, such as the rifamycin SV produced in 1961. In clinical application.
  • the nucleus of rifamycin SV is a macrocyclic lactam containing 27 carbon atoms. The ring contains a naphthalene ring.
  • naphthalene ring is connected to the C atoms of C-2 and C-12 by an aliphatic chain. It constitutes the "Ansha Bridge” structure.
  • rifamycin derivatives were synthesized using rifamycin SV as the nucleus.
  • rifampicin is currently the most widely used, chemical name 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin, molecular formula C 43 H 58 N 4 O 12 is used for the treatment of a variety of bacterial infectious diseases. It is particularly effective for tuberculosis and is the first-line drug for the treatment of tuberculosis.
  • Rifapentine is a new long-acting rifamycin antibiotic with a chemical name of 3-[[(4-cyclopentyl-1-piperazinyl)imino]methyl]-rifamycin,
  • the molecular formula is C 47 H 64 N 4 O 12 , which has a good antibacterial effect on Mycobacterium tuberculosis.
  • rifampicin which is widely used clinically, its antibacterial spectrum is similar, but its anti-tuberculosis effect is 2-10 times higher, with fewer adverse reactions and longer elimination half-life in plasma.
  • Other rifamycin antibiotics also include rifaximin, rifamycin, and rifabutin.
  • Yellow fever virus belongs to the flavivirus genus of the flaviviridae family. It is an enveloped RNA virus that is transmitted by mosquito bites. The virus can invade liver, kidney, spleen, heart, bone marrow, lymph nodes and other tissues and organs, causing a wide range of tissues. Lesions, the pathological changes of the liver have the most diagnostic specificity. Yellow fever caused by yellow fever virus infection has always been a severe infectious disease with a high incidence in South America, Africa and other countries, with a fatality rate of 30-40%. There are attenuated vaccines that can prevent yellow fever virus infection, but there are no effective specific antiviral drugs for the treatment of infected persons.
  • the present invention aims to provide a new use of rifamycin antibiotics.
  • the invention provides the application of rifamycin antibiotics in the preparation of anti-yellow fever virus infection drugs.
  • the application of the present invention is characterized in that: the above-mentioned drug refers to a semi-synthetic or synthetic broad-spectrum antibiotic containing a rifamycin core.
  • the amount of the semi-synthetic or synthetic broad-spectrum antibiotics containing the rifamycin nucleus in the medicine varies from 10 -6 to 100% by mass percentage of the component according to the difference of the dosage form and the like.
  • the above-mentioned semi-synthetic or synthetic broad-spectrum antibiotic containing rifamycin nucleus can be selected from any broad-spectrum antibacterial effect, and its inhibitory effect on gram-positive bacteria such as tuberculosis, leprosy, streptococcus, pneumococcus, etc. They are all strong, rifamycin antibiotics that can effectively inhibit certain Gram-negative bacteria.
  • the above-mentioned semi-synthetic or synthetic broad-spectrum antibiotics containing rifamycin nucleus can be selected from: rifamycin SV, a variety of rifamycin derivatives synthesized using rifamycin SV as the nucleus Substances, such as: 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin, 3-[[(4-cyclopentyl-1-piperazinyl) Amino]methyl]-rifamycin, rifaximin, rifamycin, rifabutin, etc., or derived from rifamycin B through biological/chemical reaction Substances, such as: Rifamycin O and so on.
  • the application of the present invention is characterized in that the above-mentioned drugs include one or more broad-spectrum antibiotics of the rifamycin family.
  • the amount of the above-mentioned broad-spectrum antibiotics of the rifamycin family in the medicine varies from 10 -6 to 100% by mass percentage of the component according to the difference of the dosage form and the like.
  • the application of the present invention is characterized in that the above-mentioned medicine is used to prevent or treat yellow fever virus infection.
  • the application of the present invention is characterized in that the dosage form of the above-mentioned drug is a dosage form for administration through the gastrointestinal tract.
  • the application of the present invention is characterized in that the dosage form of the above-mentioned drug is selected from powders, tablets, granules, capsules, solutions, emulsions, and suspensions.
  • the application of the present invention is characterized in that the dosage form of the above-mentioned drug is a dosage form through a route other than the gastrointestinal tract.
  • the application according to the present invention is characterized in that the dosage form of the drug administration route is selected from injection dosage form, respiratory tract dosage form, nose drops, skin dosage form, mucosal dosage form or cavity dosage form.
  • BHK cells are infected with yellow fever virus, and rifapentin, rifampicin or solvent DMSO are added at the same time, or not infected with yellow fever virus (without virus), CCK8 reagent is added 72 hours later, and the absorbance at 450 nm is detected.
  • Huh7 cells were treated with rifapentine, rifampicin and solvent DMSO respectively, and CCK8 reagent was added 72 hours later, and the absorbance at 450 nm was detected.
  • Figure 6 Diagram of the effect of rifapentine administration on the death of mice caused by yellow fever virus infection.
  • rifamycin antibiotics used in the embodiments of the present invention can all be purchased through commercial methods.
  • Virus Yellow fever virus, which was isolated from the plasma of a migrant worker in Fujian province imported into China from Angola in Africa by the Department of Biomedical Protection of the Naval Military Medical University of the Chinese People's Liberation Army in 2016, and was expanded and cultured by baby hamster kidney BHK cells.
  • Human liver cancer cell line Huh7 and baby hamster kidney BHK cells were purchased from Shanghai Institute of Cells, Chinese Academy of Sciences and preserved by the Department of Biomedical Protection, Naval Medical University of the Chinese People's Liberation Army.
  • DMEM cell culture medium is the product of Hyclone Company of the United States. When used, 10% fetal bovine serum, non-essential amino acids, ampicillin and streptomycin (100U/ml each) are added. The medium additives are all products of Thermo Fisher Company of the United States.
  • CCK8 cell viability and proliferation detection kit is a product of MedChemExpress, USA.
  • Mouse anti-yellow fever virus polyclonal antibody was prepared by immunizing mice with formaldehyde-inactivated yellow fever virus by the Department of Biomedical Protection of Naval Military Medical University of the People's Liberation Army of China.
  • Fluorescein Alexa Fluor 488-labeled anti-mouse IgG is a product of Thermo Fisher, USA.
  • Subculture baby hamster kidney BHK cells in a T75 cell culture flask with complete DMEM medium inoculate them in a 96-well plate with 10,000 cells per well, 100 ⁇ L DMEM medium, and culture for 12 hours. Then add 50 ⁇ L of complete DMEM medium containing 1000PFU (plaque forming unit) yellow fever virus to each well; at the same time add 50 ⁇ L of complete DMEM medium containing FDA small molecule chemical drugs, the final concentration of the drug is 10 ⁇ M, each concentration repeats 3 wells, Add an equal volume of solvent DMSO as a control without adding drugs. Placed in 37°C, 5% CO 2 incubator for culture. After 72 hours, all the cells in the wells with DMSO can be seen round or fall off under the microscope.
  • solvent DMSO as a control without adding drugs
  • the cultured human hepatoma cell line Huh7 and baby hamster kidney BHK cells were respectively inoculated into a 96-well plate with 10,000 cells per well and 100 ⁇ L of culture medium. After 12 hours, the original culture medium was aspirated, and a concentration gradient dilution was added to each well. 100 ⁇ L of complete DMEM medium of Fampicin or Rifapentine, the final concentrations of rifampicin and rifapentine are 2.5, 5, 10, 20, 40 and 80 ⁇ M, each concentration is repeated 3 wells, with 80 ⁇ M drug solvent The content of DMSO was used as a control without drugs. Placed in 37°C, 5% CO 2 incubator for culture.
  • Figure 2 shows the absorbance at 450 nm of Huh7 cells treated with rifampicin and rifapentine and cells treated with DMSO solvent at 80 ⁇ M.
  • the main target organ of yellow fever virus infection in the human body is the liver. Therefore, we will mainly use Huh7 cells derived from human liver cancer as target cells to observe the effect of rifamycin antibiotics on yellow fever virus infection.
  • the subcultured human liver cancer cell line Huh7 was seeded in a 96-well plate with 10,000 cells per well, 100 ⁇ L of culture medium, and cultured for 12 hours.
  • rifamycin antibiotics In order to observe whether other rifamycin antibiotics can also have anti-yellow fever virus activity, we further carried out a quantitative test of anti-yellow fever virus activity of five rifamycin antibiotics, that is, by detecting the antiviral activity at different concentrations Activity, calculate the half inhibitory concentration (IC50) of the drug against the virus.
  • the five rifamycin antibiotics include rifampicin, rifapentin, rifaximin, rifamycin, and rifabutin.
  • concentration gradients of 0.2, 1, 5, and 25 ⁇ M which were successively diluted five-fold, were set for each drug. In the detection method, except that the concentration gradient is set for the drug treatment, the others are the same as those described in (3).
  • the cell imaging and analysis system is used to take pictures of the cells. Four fields of view are taken in each hole to analyze and calculate the percentage of green fluorescent positive cells, that is, the virus infection rate (as shown in the figure) 4), according to the percentage of positive cells in the wells of each concentration gradient drug treatment, the IC50 of the five rifamycin antibiotics was calculated: rifampicin IC50: 1.964 ⁇ M; rifapentine IC50: 0.347 ⁇ M; Ming IC50: 3.246 ⁇ M; Rifamycin: 14.125 ⁇ M; Rifabutin: 24.547 ⁇ M. Among them, rifapentine has the lowest IC50 and shows the strongest anti-yellow fever virus activity.
  • Rifapentine is a new long-acting rifamycin antibiotic. Its antibacterial activity against Mycobacterium tuberculosis is significantly better than that of rifampicin. After oral administration, it is widely distributed in body tissues and body fluids with a blood elimination half-life of 14-18 hours. In the previous part of the experiment, we calculated that when rifapentine acts on Huh7 cells, the activity of inhibiting yellow fever virus is the strongest. Therefore, we further observed the anti-yellow fever virus activity of rifapentine in mice.
  • mice We first conducted a preliminary study on the dose of virus challenge in mice, and infected mice by intraperitoneal injection at a dose of 1.8*10 8 PFU. The mice died on the 4th day after the injection, and all the mice died on the 6th day. This dose is a relatively high lethal dose.
  • Rifapentine dry powder is initially dissolved in DMSO, and then fully dissolved in PBS containing 40% PEG300-5% Tween 80, and administered by gavage at a dose of 40 mg/Kg/day, once a day.
  • a total of 42 female C57BL/6 mice at the age of 6 weeks were randomly divided into four groups: 1. Early administration group. The administration was started 2 days before the virus attack, and the intragastric administration was given on the day of the virus attack, followed by intraperitoneal administration. Virus injection, 13 mice; 2. Post-administration group, intragastric administration 24 hours after virus attack, followed by intragastric administration every day, 13 mice; 3.
  • DMSO group intragastric drug administration 24 hours after virus attack Solvent, followed by intragastric administration of drug solvent every day, 13 mice; 4. Blank control group, no virus challenge, no administration, 4 mice. Virus attack was carried out by intraperitoneal injection, with a dose of 1.8*10 8 PFU yellow fever virus.
  • mice Weigh the mice from the day of the virus attack (before the intraperitoneal injection of the virus), and weigh the mice every day before gavage. Observe the survival of the mice twice a day (during the gavage and 12 hours after the gavage) Two observations), record the changes in the weight and survival of the mice.
  • mice The weight changes of mice are shown in Figure 5: the recorded weight of the DMSO group began to decrease from the fourth day after the virus challenge, and the recorded weight of the mice in the early administration group and the post-administration group also began on the fourth day after the virus challenge. Decrease, but the weight of the surviving mice began to increase on the eighth day after the virus challenge.
  • the results show that rifapentine can effectively resist yellow fever virus infection in mice, and has the effect of preventing and treating yellow fever virus infection.

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Abstract

涉及医药技术领域，关于利福霉素类抗生素在制备抗黄热病毒感染药物中的应用。利福霉素类抗生素是指含有利福霉素衍生物的半合成或合成抗生素包括利福霉素家族的多种广谱抗生素，用于治疗细菌感染，疗效肯定，副作用低，安全性好。

Description

利福霉素类抗生素在制备抗黄热病毒感染药物中的应用 技术领域
本发明属于医药技术领域，具体涉及利福霉素类抗生素在制备抗黄热病毒感染药物中的应用。
背景技术
利福霉素类抗生素是指含有利福霉素衍生物的半合成或合成抗生素，包括利福霉素家族的多种抗生素，具有广谱抗菌作用，对结核杆菌、麻风杆菌、链球菌、肺炎球菌等革兰氏阳性细菌的抑制作用都很强，对某些革兰氏阴性菌也能有效抑制。
天然的利福霉素类抗生素最初是意大利一药厂从地中海链霉菌培养液中分离出来，其中的利福霉素B活性较强，性质较稳定，但临床药效不够理想。后经人工对天然产的利福霉素B作各种化学结构的改变，得到一系列药效更强的抗生素，称为半合成利福霉素，如1961年生产的利福霉素SV首先在临床应用。利福霉素SV母核是一个含有27个碳原子的大环内酰胺，环中含一个萘环，萘环上，由一脂肪链分别与C-2和C-12的C原子相连接，构成“安莎桥”结构。后又以利福霉素类SV为母核合成了多种利福霉素类衍生物。其中，利福平(rifampicin)是目前应用最广泛的一种，化学名3-[[(4-甲基-1-哌嗪基)亚氨基]甲基]-利福霉素，分子式C 43H 58N 4O 12，用于多种细菌感染性疾病的治疗，对结核病的疗效尤为突出，是治疗结核病的第一线药物。利福喷汀(rifapentine)，是新型长效利福霉素类抗生素，化学名3-[[(4-环戊基-1-哌嗪基)亚氨基]甲基]-利福霉素，分子式C 47H 64N 4O 12，对结核杆菌具有良好的抗菌作用。与临床应用广泛的一线抗结核药利福平相比，其抗菌谱相似，但抗结核杆菌作用要高2-10倍，并且不良反应少，血浆中消除半衰期更长。其他的利福霉素类抗生素还包 括利福昔明(Rifaximin)、利福霉素(Rifamycin)、利福布汀(Rifabutin)等。
黄热病毒(yellow fever virus)属于黄病毒科黄病毒属，为有包膜的RNA病毒，经蚊虫叮咬传播，病毒可侵犯肝、肾、脾、心、骨髓和淋巴结等组织器官，引起广泛组织病变，其中肝脏病理变化最具诊断的特异性。黄热病毒感染引起的黄热病一直是南美、非洲等国家高发的烈性传染病，病死率高达30-40％。有减毒疫苗可预防黄热病毒感染，但无有效特异性抗病毒药物用于感染者治疗。
发明内容
本发明旨在提供一种利福霉素类抗生素的新用途。
本发明提供了，利福霉素类抗生素在制备抗黄热病毒感染药物中的应用。
本发明所述之应用，其特征在于：上述药物是指含有利福霉素母核的半合成或合成广谱抗生素。上述含有利福霉素母核的半合成或合成广谱抗生素在药物中的添加量，根据剂型等的差异，该组分占药物质量百分比含量10 -6-100％不等。
优选地，上述含有利福霉素母核的半合成或合成广谱抗生素可以选自任何具有广谱抗菌作用，对结核杆菌、麻风杆菌、链球菌、肺炎球菌等革兰氏阳性细菌的抑制作用都很强，对某些革兰氏阴性菌也能有效抑制的利福霉素类抗生素。
更优选地，上述含有利福霉素母核的半合成或合成广谱抗生素可以选自：利福霉素SV，以利福霉素类SV为母核合成了多种利福霉素类衍生物,如:3-[[(4-甲基-1-哌嗪基)亚氨基]甲基]-利福霉素，3-[[(4-环戊基-1-哌嗪基)亚氨基]甲基]-利福霉素，利福昔明(Rifaximin)、利福霉素(Rifamycin)、利福布汀(Rifabutin)等，或者由利福霉素B经生物/化学反应获取的衍生物，如： 利福霉素O等。
本发明所述之应用，其特征在于：上述药物包括利福霉素家族的一种或多种广谱抗生素。上述利福霉素家族的广谱抗生素，在药物中的添加量，根据剂型等的差异，该组分占药物质量百分比含量10 -6-100％不等。
本发明所述之应用，其特征在于：上述药物用于预防或治疗黄热病毒的感染。
本发明所述之应用，其特征在于：上述药物的剂型为经胃肠道给药剂型。
本发明所述之应用，其特征在于：上述药物的给药剂型选自散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂。
本发明所述之应用，其特征在于：上述药物的剂型为经胃肠道以外给药途径剂型。
本发明所述之应用，其特征在于：上述药物的给药途径剂型选自注射给药剂型、呼吸道给药剂型、滴鼻剂、皮肤给药剂型、黏膜给药剂型或腔道给药剂型。
附图说明
图1.利福喷汀与利福平以及溶剂DMSO保护BHK细胞抵抗黄热病毒感染的结果对比图；
即、BHK细胞以黄热病毒感染，同时加入利福喷汀、利福平或溶剂DMSO，或不以黄热病病毒感染(未加病毒)，72小时后加入CCK8试剂，检测450nm吸光值。
图2.利福喷汀、利福平以及溶剂DMSO处理的Huh7细胞的活性对比图；
即、以利福喷汀、利福平以及溶剂DMSO分别处理Huh7细胞，72小时后加入CCK8试剂，检测450nm吸光值。
图3.利福平、利福喷汀与溶剂DMSO在Huh7细胞感染模型中对黄热病毒的抑制作用对照图。
图4.五种利福霉素类抗生素抑制黄热病毒感染的效果对比图。
图5.给药利福喷汀对黄热病毒感染引起小鼠体重变化的影响图。
图6.给药利福喷汀对黄热病毒感染致小鼠死亡的影响图。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
本发明实施例所用的利福霉素类抗生素均可以通过市售方式购买获得。
一、病毒、药物、试剂及其他材料
1.病毒：黄热病毒，由中国人民解放军海军军医大学生物医学防护教研室于2016年从一例由非洲安哥拉输入中国的福建省务工人员血浆中分离出，经幼仓鼠肾BHK细胞扩增培养。
2.化合物：978种美国FDA化学药物分子库，购自美国Selleck公司。
3.人肝癌细胞系Huh7和幼仓鼠肾BHK细胞，购自中国科学院上海细胞所，由中国人民解放军海军军医大学生物医学防护教研室保存。
4.DMEM细胞培养液为美国Hyclone公司产品，用时添加10％胎牛血清、非必需氨基酸、氨苄青霉素和链霉素(各100U/ml)，培养液添加剂均为美国Thermo Fisher公司产品。
5.细胞消化液，含0.25％胰蛋白酶，用磷酸盐缓冲液配制。
6.CCK8细胞活性和增殖检测试剂盒为美国MedChemExpress公司产品。
7.小鼠抗黄热病毒多克隆抗体由中国人民解放军海军军医大学生物医学防护教研室用甲醛灭活的黄热病毒免疫小鼠而制备。
8.荧光素Alexa Fluor 488-标记的抗小鼠IgG为美国Thermo Fisher公司产品。
二、实验方法：
(一)从含978个小分子化合物的FDA药物小分子库中筛选抗黄热病毒药物
用完全DMEM培养液在T75细胞培养瓶内传代培养幼仓鼠肾BHK细胞，接种于96孔板，每孔10000个细胞，DMEM培养液100μL，培养12小时。随后每孔加入50μL含1000PFU(空斑形成单位)黄热病毒的完全DMEM培养液；同时加入50μL含FDA小分子化学药物的完全DMEM培养液，药物终浓度为10μM，每个浓度重复3孔，以加等体积溶剂DMSO作为不加药物的对照。置于37℃、5％CO 2孵箱内培养。72小时后显微镜下可见加DMSO孔的全部细胞均变圆或脱落。每孔加入CCK8细胞活性和增殖检测试剂10μL，置于37℃、5％CO 2孵箱内，30分钟后用多功能酶标仪检测每孔在450nm波长的吸光值，计算药物对细胞的保护率＝(加药细胞450nm吸光值—加DMSO孔细胞450nm吸光值)/未加病毒加DMSO的细胞450nm吸光值*100％，得到每种药物在10μM浓度时对黄热病毒感染细胞的保护率。结果显示，两种利福霉素类抗生素利福平与利福喷汀对细胞具有显著的保护效果，两种抗生素以及DSMO处理细胞的450nm吸光值如图1所示，计算得到的利福平与利福喷汀的细胞保护率分别为81.5％和94.1％。
(二)利福平与利福喷汀对细胞的毒性
分别将培养的人肝癌细胞系Huh7和幼仓鼠肾BHK细胞接种于96孔板，每孔10000个细胞，培养液100μL，12小时后，吸除原培养液，每孔内加入浓度 梯度稀释的利福平或利福喷汀的完全DMEM培养液100μL，利福平与利福喷汀终浓度分别为2.5、5、10、20、40和80μM，每个浓度重复3孔，以80μM药物的溶剂DMSO含量作为不加药物的对照。置于37℃、5％CO 2孵箱内培养。48小时后，每孔加入CCK8细胞活性和增殖检测检测试剂10μL，置于37℃、5％CO 2孵箱内，30分钟后用多功能酶标仪检测每孔对450nm波长的吸光值，根据不同浓度药物处理孔与溶剂孔450nm吸光值的差异评价药物的细胞毒性。
结果显示，当浓度等于或低于80μM时，利福平与利福喷汀处理的两种细胞与DMSO溶剂处理的细胞均无明显差异。图2显示80μM时，利福平与利福喷汀处理的Huh7细胞与DMSO溶剂处理细胞的450nm吸光值。
(三)利福平与利福喷汀在细胞感染模型中对黄热病毒的抑制作用
黄热病毒在人体内主要感染的靶器官是肝脏，因此，我们后续主要用人肝癌来源的Huh7细胞为靶细胞，观察利福霉素类抗生素对黄热病毒感染的影响。将传代培养的人肝癌细胞系Huh7接种于96孔板，每孔10000个细胞，培养液100μL，培养12小时。随后每孔加入50μL含1000PFU黄热病毒的完全DMEM培养液；同时加入50μL含利福平或利福喷汀的完全DMEM培养液，药物终浓度为5μM，每个浓度重复3孔，以加等体积溶剂DMSO作为不加药物的对照。置于37℃、5％CO 2孵箱内培养。
20小时后，用免疫荧光技术检测病毒对细胞的感染情况，具体操作如下：吸除培养板中的培养液，每孔加100μL甲醇，将培养板至于-20℃冰箱，20分钟后，取出培养板，吸除甲醇，每孔以磷酸盐缓冲液(PBS)洗孔一次，随后加入100μL含3％牛血清白蛋白(BSA)的PBS(以下简称3％BSA-PBS)，置于水平摇床上，室温缓慢摇1小时，吸除培养板中的3％BSA-PBS，每孔加100μL含抗黄热病毒多克隆抗体的1％BSA-PBS(抗体500倍稀释)，室温缓慢摇1小时，吸 除培养板中的抗黄热病毒多克隆抗体工作液，每孔以PBS洗3次，随后加入100μL含荧光素Alexa Fluor 488-标记的抗小鼠IgG的1％BSA-PBS(荧光素抗体1500倍稀释)，室温避光缓慢摇1小时，吸除培养板中的荧光素抗体工作液，每孔加入DAPI细胞核染色液100μL，室温避光缓慢摇10分钟，吸除培养板中的DAPI细胞核染色液，每孔以PBS洗3次，用细胞成像及分析系统(BioTek Cytation 5 Imaging Reader)对每孔细胞的荧光分布进行拍照。
结果如图3所示，与作为对照的DMSO溶剂处理孔相比，利福平处理孔仅可见到少数的绿色荧光细胞，而利福喷汀处理孔未见到明确的绿色荧光细胞。结果证明，在1μM浓度时，这两种药物均能显著抑制黄热病毒对Huh7细胞的感染。
(四)利福霉素类抗生素抑制黄热病毒感染活性的定量测定
为了观察其他利福霉素类抗生素是否也能具有抗黄热病毒活性，我们进一步对五种利福霉素类抗生素进行了抗黄热病毒活性的量化检测，即通过检测不同浓度时的抗病毒活性，计算药物对病毒的半数抑制浓度(IC50)。五种利福霉素类抗生素包括利福平、利福喷汀、利福昔明、利福霉素、利福布汀。每种药物设置了连续五倍稀释的四个浓度梯度0.2、1、5、25μM。检测方法中，除了药物处理设置浓度梯度外，其他与(三)中所述相同。病毒感染的细胞经病毒抗体和荧光抗体结合以及细胞核染色以后，用细胞成像及分析系统进行细胞拍照，每孔拍照四个视野，分析、计算绿色荧光阳性细胞的百分率，即病毒感染率(如图4所示)，根据各浓度梯度药物处理孔的阳性细胞百分率，计算出五种利福霉素类抗生素的IC50：利福平IC50：1.964μM；利福喷汀IC50：0.347μM；利福昔明IC50：3.246μM；利福霉素：14.125μM；利福布汀：24.547μM。其中利福喷汀的IC50最低，显示出最强的抗黄热病毒活性。
(五)利福喷汀能有效保护小鼠抵抗黄热病毒感染，降低小鼠死亡率
利福喷汀是新的长效利福霉素类抗生素，对结核杆菌的抗菌活性显著优于利福平，口服后广泛分布于全身组织及体液，血消除半衰期达14-18小时。在上一部分实验中，我们测算出利福喷汀作用于Huh7细胞时，抑制黄热病毒的活性最强。因此，我们进一步观察了利福喷汀在小鼠体内的抗黄热病毒活性。
我们先对小鼠进行了病毒攻毒剂量的初步研究，以1.8*10 8PFU剂量腹腔注射感染小鼠，在注射以后的第4天即有小鼠死亡，第6天小鼠全部死亡，显示该剂量是一个相对较高的致死剂量。
利福喷汀干粉先以DMSO初步溶解，然后以含40％PEG300-5％Tween 80的PBS充分溶解，以灌胃方式给药，剂量40mg/Kg/天，每天一次。6周龄、雌性C57BL/6小鼠共42只，随机分为四组，分别为：1、提前给药组，病毒攻击以前2天开始给药，病毒攻击当天先灌胃给药，随后腹腔注射病毒，13只小鼠；2、后给药组，病毒攻击24小时以后灌胃给药，随后每天灌胃给药，13只小鼠；3、DMSO组，病毒攻击24小时以后灌胃药物溶剂，随后每天灌胃药物溶剂，13只小鼠；4、空白对照组，不进行病毒攻击，也不给药，4只小鼠。以腹腔注射方式进行病毒攻击，剂量1.8*10 8PFU黄热病毒。
从病毒攻击当天(在腹腔注射病毒前)开始称小鼠体重，此后每天在灌胃给药前称小鼠体重，每天两次观察小鼠存活情况(灌胃给药时以及给药12小时以后两次观察)，记录小鼠体重和生存的变化情况。
小鼠体重变化如图5所示：DMSO组体重记录数值从病毒攻击后的第四天开始降低，提前给药组和后给药组小鼠体重记录数值也是从病毒攻击后的第四天开始降低，但存活小鼠的体重在病毒攻击后第八天开始呈上升趋势。
小鼠生存情况如图6所示：DMSO组从病毒攻击后第四天开始有小鼠死亡， 至第六天，13只小鼠全部死亡；提前给药组和后给药组也在病毒攻击以后第四天开始有小鼠死亡，但相比DMSO组，小鼠死亡时间相对滞后，且在病毒攻击以后第七天至观察结束时，两组分别有4只和3只小鼠一直存活。结果显示：利福喷汀在小鼠体内能有效抗黄热病毒感染，起到预防和治疗黄热病毒感染的效果。
上述体外和体内实验结果均表明，利福霉素类抗生素具有显著的抗黄热病毒感染的活性，可用于制备抗黄热病毒感染的药物，用于黄热病毒感染的预防和治疗。
以上显示和描述了本发明的主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。

Claims (8)

  1. 利福霉素类抗生素在制备抗黄热病毒感染药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述利福霉素类抗生素是指含有利福霉素母核的半合成或合成广谱抗生素。
  3. 根据权利要求1所述的应用，其特征在于：所述药物包括利福霉素家族的一种或多种广谱抗生素。
  4. 根据权利要求1-3任一所述的应用，其特征在于：所述药物用于预防或治疗黄热病毒的感染。
  5. 根据权利要求1-4任一所述的应用，其特征在于：所述药物的剂型为经胃肠道给药剂型。
  6. 根据权利要求1-4任一所述的应用，其特征在于：所述药物的给药剂型选自散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂。
  7. 根据权利要求1-4任一所述的应用，其特征在于：所述药物的剂型为经胃肠道以外给药途径剂型。
  8. 根据权利要求1-4任一所述的应用，其特征在于：所述药物的给药途径剂型选自注射给药剂型、呼吸道给药剂型、滴鼻剂、皮肤给药剂型、黏膜给药剂型或腔道给药剂型。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115006386A (zh) * 2022-03-28 2022-09-06 中国人民解放军海军军医大学 硝呋齐特在制备抗蜱传脑炎病毒西尼罗病毒黄热病毒和基孔肯雅热病毒感染药物中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1080853A (zh) * 1992-03-18 1994-01-19 美国生物科学有限公司 N-(膦酰基乙酰基)-l-天冬氨酸组合物及其作为广谱抗病毒的使用方法
CN101395259A (zh) * 2005-12-14 2009-03-25 活跃生物药物学有限公司 利福霉素类似物及其应用
WO2013026994A1 (en) * 2011-08-24 2013-02-28 Isis Innovation Limited Mosquitoes with enhanced pathogen resistance
CN104622927A (zh) * 2014-12-27 2015-05-20 江传福 外用消炎粉
CN105287427A (zh) * 2015-10-20 2016-02-03 沈阳红旗制药有限公司 一种利福平半固体胶囊及其制备方法
CN105878246A (zh) * 2015-01-22 2016-08-24 李慧 一种半合成抗生素

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1080853A (zh) * 1992-03-18 1994-01-19 美国生物科学有限公司 N-(膦酰基乙酰基)-l-天冬氨酸组合物及其作为广谱抗病毒的使用方法
CN101395259A (zh) * 2005-12-14 2009-03-25 活跃生物药物学有限公司 利福霉素类似物及其应用
WO2013026994A1 (en) * 2011-08-24 2013-02-28 Isis Innovation Limited Mosquitoes with enhanced pathogen resistance
CN104622927A (zh) * 2014-12-27 2015-05-20 江传福 外用消炎粉
CN105878246A (zh) * 2015-01-22 2016-08-24 李慧 一种半合成抗生素
CN105287427A (zh) * 2015-10-20 2016-02-03 沈阳红旗制药有限公司 一种利福平半固体胶囊及其制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115006386A (zh) * 2022-03-28 2022-09-06 中国人民解放军海军军医大学 硝呋齐特在制备抗蜱传脑炎病毒西尼罗病毒黄热病毒和基孔肯雅热病毒感染药物中的应用
CN115006386B (zh) * 2022-03-28 2024-03-26 中国人民解放军海军军医大学 硝呋齐特在制备抗蜱传脑炎病毒西尼罗病毒黄热病毒和基孔肯雅热病毒感染药物中的应用

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