CN100430061C使用内过氧化物治疗黄病毒科引起的感染，包括丙型肝炎、牛病毒性腹泻和经典猪瘟病毒---内过氧化物和青蒿素更具体地具有治疗丙型肝炎病毒感染的功效｜CN100430061C Use of peroxides to treat infections caused by Flaviviridae, including hepatitis C, bovine viral diarrhea, and classical swine fever viruses--- Peroxides and a
Entry posted by Roger in Artemisinin Patent｜青蒿素专利
77 views
CN100430061C使用内过氧化物治疗黄病毒科引起的感染，包括丙型肝炎、牛病毒性腹泻和经典猪瘟病毒---内过氧化物和青蒿素更具体地具有治疗丙型肝炎病毒感染的功效
CN100430061C Use of peroxides to treat infections caused by Flaviviridae, including hepatitis C, bovine viral diarrhea, and classical swine fever viruses--- Peroxides and artemisinin specifically have the effect of treating hepatitis C virus infection.
内过氧化物在制备治疗由黄病毒科病毒(包括丙型肝炎病毒、牛病毒性腹泻病毒和猪瘟病毒)引起的感染的药物中的应用
倍半萜类，特别是倍半萜内酯内过氧化物，例如青蒿素及其类似物，在治疗丙型肝炎病毒感染中的应用。对青蒿素、青蒿素的类似物和一些蒿的粗提取物进行了体外抗DNA病毒、逆转录病毒和黄病毒科病毒(一种重要的人和动物RNA病原体科)的测试。筛选了这些化合物的抗肿瘤活性。观察到青蒿素具有抗牛病毒性腹泻病毒的强活性。由于瘟病毒(如BVDV)与丙型肝炎病毒(HCV)具有许多相似性，我们能够得出结论，在治疗丙型肝炎病毒感染方面，内过氧化物具有一般的效果，青蒿素具有更特异的效果。
内过氧化物在制备治疗由黄病毒科病毒(包括丙型肝炎病毒、牛病毒性腹泻病毒和猪瘟病毒)引起的感染的药物中的应用
发明背景
本发明大体上涉及倍半萜内酯类(sesquiterpene lactones)在治疗由黄病毒科(Flaviviridae)引起的感染中的应用，特别是倍半萜内酯内过氧化物在治疗丙型肝炎感染、黄热病、登革热、牛病毒性腹泻和猪瘟(classical swine fever)中的应用。
慢性丙型肝炎病毒感染是一个影响全世界1.8亿人(总人口的3％)的一个重要的公共健康问题，这里面包括美国的4百万人，而且是导致肝慢性病的一个主要原因。有人预期美国的HCV感染将继续增加，到2010年感染者将达到现在的3倍。丙型肝炎病毒的感染可能引起严重的，在一些病例中可能是威胁生命的慢性肝病，包括肝衰竭和肝癌。
在美国，慢性肝病是导致成人死亡的第十位原因，并且导致8每年接近25000例或接近1％的死亡人数。人口基础上的研究表明：40％的慢性肝病是HCV相关的，导致每年大约8000-10000的死亡。HCV相关的急性和慢性肝病的医疗和工作损失估计每年要超过6亿美元。并且HCV相关的晚期肝病是成人肝移植最常见的原因。因为大多数的HCV感染者的年龄在30-49岁之间，由与HCV相关的慢性肝病引起的死亡数可能在未来的10-20年间大幅增加，这时候这组感染者将达到慢性肝病的并发症通常发生的年龄。
HCV的传播主要是通过皮肤与血的大量或重复的直接接触。在美国，与HCV传播有关的两种最常见的接触是输血和注射毒品。丙型肝炎的治疗是临床实践中变化快速的一个领域。干扰素和利巴韦林(一种核酸类似物)的联合治疗已被批准用于慢性丙型肝炎的首次治疗。对应用利巴韦林和干扰素的联合治疗的患者的研究证实，与单独应用干扰素治疗的反应率15-25％相比，其持久的反应率有显著增加，达到40-50％。多数患者在治疗早期有干扰素一过性感冒样症状，但是这些症状在继续治疗中减轻。随后的副作用有疲劳、骨髓抑制和中枢精神作用(例如，淡漠、认知变化、暴躁和抑郁)。由于严重的副作用，10-40％的患者的干扰素的剂量必须减少，5-15％的患者必须间断使用。利巴韦林能引起溶血性贫血，并且对于患者原来存在的贫血、骨髓抑制或肾衰竭来说也是有问题的。在这些患者，应避免联合治疗，或应该试图纠正贫血。利巴韦林引起的溶血性贫血对有缺血性心脏疾病或脑血管疾病的患者也可能是威胁生命的。利巴韦林是引起畸形的，女性患者在治疗过程中要避免妊娠。其他治疗，包括皮质类固醇、脱氧熊胆酸(ursodiol)和胸腺素，是无效的。肝脏的高铁水平可降低干扰素的药效。去铁治疗(静脉切开放血术或螯合术)与干扰素的联合应用已经研究了，但是，还尚无结论(www.cdc.gov)。所以，在医学领域迫切需要发现和发展可以用来治疗HCV感染、同时具有较少或者能够减少副作用的、并且与现有的治疗相比有更好疗效的新的生物活性分子。
发明内容
已经发现具有以下通式的倍半萜内酯：
具有对抗由黄病毒科，包括丙型肝炎病毒、牛病毒性腹泻病毒和猪瘟病毒引起的感染的活性。本申请描述了一些具有代表性的、目前优选的倍半萜内酯类，不过，本领域人员显然能够理解，公知技术中其他倍半萜内酯化合物在由黄病毒科引起的感染的治疗中也是有效的。还包括此类化合物的药学上可接受的盐。
优先选用的倍半萜内酯是倍半萜内酯内过氧化物青蒿素(artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(artensunate)和蒿甲醚(artemether)。
附图说明
图1是对青蒿素、双氢青蒿素、青蒿琥酯和蒿甲醚化学结构的图解说明。
具体实施方式
本申请中，我们将介绍倍半萜内酯类的抗病毒效应，特别是倍半萜内酯内过氧化物，例如青蒿素、双氢青蒿素、青蒿琥酯和蒿甲醚对特定的黄病毒科病毒例如丙型肝炎病毒的抗病毒作用。
本发明的优选的倍半萜化合物包含具有以下通式的化合物：
其中：X1和X2选自O、S、Se和NH；Y选自O、S、Se和NH；Z选自O、NH、S和Se，且Q选自CO、CHOH、CHOCH3、CHOC2H5、CHOC3H7和CHOCOCCH2CH2COOH及其药物可接受盐。
本发明特别优选的倍半萜化合物包括青蒿素，其X1和X2是O、Y是O、Z是O、并且Q是C＝O；双氢青蒿素(同青蒿素，但Q为CHOH)；蒿甲醚(同青蒿素，但Q为CHOCH3)；蒿乙醚(arteether)(同青蒿素，但Q为COC2H5)；一种丙基产品(同青蒿素，但Q为CHOC3H7)；及青蒿琥酯(artesunate)(同青蒿素，但Q为CHOCOCH2CH2COOH)。本发明的最优选的倍半萜化合物是双氢青蒿素。
内过氧化物有一个二氧键(peroxo linkage)(-O-O)，这个结构被认为对这些产物具有抗疟疾活性是很重要的。用-S-S-(二硫键)，或-Se-Se-(二硒键)，或-N-O-或-NH-NH-(肼)，以及上述键的不同组合，会产生新的化合物，也可能具有活性。
黄病毒科是人类和动物一个重要的RNA病毒病原体科(Rice CM.1996.Flaviviridae：the viruses and their replication.In：Fields BN，Knipe DM，Howley PM，eds.Fields virology.Philadelphia：Lippincott-Raven Publishers.Pp 931-960)。目前已发现的黄病毒科的属在传播途径、宿主范围和发病机理方面表现不同。典型的黄病毒成员是黄热病毒、登革病毒和瘟病毒(pestiviruses)，例如牛病毒性腹泻病毒(BVDV)和猪瘟病毒(CSFV)。该家族最新的最具特征的成员是人的常见的且专有的病原体，即丙型肝炎病毒(HCV)。黄病毒是具有(+)有义RNA基因组极性的单链RNA病毒。其他具有(+)有义RNA的病毒科包括小核糖核酸病毒科(Picornaviridae)、披膜病毒科(Togaviridae)、杯状病毒科(Caliciviridae)和冠状病毒科(Coronaviridae)。
本发明化合物可以应用它们的原形或它们的盐。在一些情况，这些化合物由足够的碱性或酸性能够形成稳定的无毒的酸或碱的盐，这些化合物以盐的形式施用是适合的。药学可接受的盐的例子是与形成的生理学可接受阴离子的有机酸形成的有机酸盐，例如抗坏血酸醋酸盐，安息香酸盐，柠檬酸盐，etoglutarate，甘油磷酸盐，丙二酸盐，甲磺酸盐，琥珀酸盐和酒石酸盐。合适的无机酸盐也可以被合成，包括重碳酸盐，碳酸盐，盐酸盐，硝酸盐，和硫酸盐。
本发明可以含有所述化合物以及合适的赋形剂的药物组合物而常规给药，该组合物对抗病毒感染是有用的。根据所治疗的是内或外病毒感染，本发明的化合物和组合物可以经胃肠外，局部，阴道内，口服或直肠给药。
对胃肠外给药，活性成分或它的盐溶液可以用水制备，任选地和无毒的表面活性剂混合。也可以在甘油，液体聚乙二醇，三乙酰甘油酯和上述试剂的混合物和在油剂里制备。
化合物可使用的剂量可以通过在体外比较其活性而决定。对人的有效量的外推方法是本领域已知的。
本化合物可以单位剂量的形式常规给药；例如每单位剂量形式包含活性成分0.1-2000mg，优选为100-1000mg，最优选为100-500mg。所需药量可以常规地一次性给药或以合适的间隔分次给药，例如每天可分2，3，4或更多次给药。每次的亚剂量还可以进一步分开，例如分成一些分散的松散分隔的施用形式；例如多次从送药器吸入或多次点眼给药。
对体内感染，组合物可以按游离药物计算的剂量水平通过口服或胃肠外给药，大约为1-30mg/kg(哺乳动物体重)，优选1-10mg/kg(哺乳动物体重)。
在此揭示的化合物和组合物的给药的精确的方案将必须依赖于被治疗的个体的需要，治疗的类型，当然还有主治医师的判定。本发明的化合物可用于有该治疗需求的动物。大多情况下，是用于人，但是家畜和宠物也可以特别地预期属于本发明的应用范围。
材料和方法
青蒿素购于Aldrich。洋艾(Artemisia absinthum)KE-4、黄花蒿(Artemisia annua)KE-5和龙蒿(Artemisia dracunculus)KE-6的植物粗提取物从美国Des Moines，Kemin工业有限公司获得。为了制备KE-4、-5和-6样品，将2g干的磨碎的苦艾、甜苦艾或龙蒿在200ml己烷中室温搅拌8小时，然后，悬浮液经过G3玻璃滤器过滤和低压下蒸发。残留的黑的滤渣用于生物筛选。蒿甲醚、双氢青蒿素和青蒿琥酯购于Dafra Pharma。上述化合物都进行了抗不同致病病毒的筛选，例如人免疫缺陷病毒(HIV)、单纯疱疹病毒(HSV)、水痘病毒(VV)、带状疱疹病毒(VZV)和人巨细胞病毒(CMV)。对所有的病毒，除了CMV外，检测了EC50(对HIV在人CEM细胞培养物中诱导的致细胞病性、HSV和VV在人胚胎成纤维细胞E6SM细胞培养物中诱导的致细胞病性、和VZV在人胚胎肺(HEL)细胞培养物中诱导的噬斑形成达到50％的抑制所需的有效化合物的浓度)。为了检测抗病毒活性，抗CMV用IC50表示，人胚胎肺成纤维(HEL)细胞用96孔板培养，每孔用20PFU病毒感染。
在37℃培养2小时后，感染的细胞中加入0.1ml含测试化合物的梯度稀释的培养液。第7天经细胞Giemsa染色后在显微镜下计数噬斑。最小抗病毒浓度以抑制病毒诱导的噬斑形成达到50％所需的剂量表示。
对这些化合物也进行了抗黄病毒的筛选。因为事实上没有足够的体外分析用于抗HCV筛选，所以决定筛选抗牛病毒性腹泻病毒(BVDV)，因为它与丙型肝炎病毒有很多相似性(Frolovl，McBride S and Rice CM.Cis-acting RNA elements required for replication of bovine.Viral diarrheavirus-hepatitis C virus 5’non-translated region chimeras.RNA 4，1418-1435(1998))。进一步对蒿甲醚抗BVDV的不同株的作用进行了筛选。
对这些化合物也进行了抗肿瘤活性的检测，通过对鼠白血病细胞(L1210/0)、鼠乳腺癌细胞(FM3A)和人T淋巴细胞(Molt4/C8，CEM/0)的增殖(的检测)进行。观察到的对CMV和VZV的最好的抗病毒活性来自KE-6。其次是青蒿素对HSV-2和VV的抗病毒活性。
结果和讨论
在第一批筛选中(见表1)，检测了青蒿素和一些蒿属粗提取物(KE-4、KE-5和KE-6)对HIV-1、HIV-2、HSV-1、HSV-2、VV、CMV和VZV的抗病毒活性。
表1：抗HIV-1、HIV-2、HSV-1、HSV-2、VV、CMV和VZV的筛选结果
a50％对HIV在人淋巴细胞CEM细胞培养物中诱导的致细胞病性、HSV和VV在人胚胎成纤维细胞E6SM细胞培养物中诱导的致细胞病性、和CMV和VZV在人胚胎肺HEL细胞培养物中诱导的噬斑形成达到50％的抑制所需的有效的化合物浓度。
另外进行了第二个筛选(表2)，以便检测在牛肾(MDBK)细胞中抗牛病毒性腹泻病毒的活性。
表2：在MDBK细胞中抗BVDV的筛选结果
抗病毒活性用BVDV的Pe515株在MDBK细胞上来评估。抗病毒活性和细胞毒性的检测都应用MTS方法。
EC50是降低病毒引起的细胞毒性效果50％时所需要的浓度。MTC(最小毒性浓度)定义为引起细胞代谢降低＞＝20％的浓度。
当用蒿甲醚、青蒿素、KE4和KE5处理MDBK细胞时，在最高浓度(100μg/ml)仍没有达到MTC。本试验中观察到内过氧化物青蒿素、双氢青蒿素、蒿甲醚和青蒿琥酯具有很强的抗黄病毒BVDV的活性，同时具有低细胞毒性。所以，这些产品可以用于治疗由黄病毒引起的感染。
以蒿甲醚进行了抗不同BVDV株，和抗BDV(边界病病毒)的筛选(表3)
表3：蒿甲醚抗BVDV和BDV的筛选结果
| 蒿甲醚 | 100μg/ml | 20μg/ml | 4μg/ml | 800ng/ml | 160ng/ml | 32ng/ml | 6.4ng/ml | 1.28ng/ml | 0.512ng/ml | 0.256ng/ml |
| 非感染 | 毒性 | |||||||||
| BVDVII ncp | 毒性 | ++ | ++ | ++ | +++ | +++ | +++ | +++ | +++ | +++ |
| BVDVI cp | 毒性 | + | + | + | + | ++ | ++ | ++ | ++ | ++ |
| BVDVI ncp | 毒性 | + | + | + | ++ | +++ | +++ | +++ | +++ | +++ |
| BDV | 毒性 | + | + | + | ++ | ++ | ++ | ++ | ++ | ++ |
+++ 所有细胞都是阳性的
++ +/-50％细胞是阳性的
+ +/-10％细胞是阳性的
BVDV 牛病毒性腹泻病毒
BDV 边界病病毒
cp 致细胞病变株
ncp 非致细胞病变株
特别地抗BVDVIcp(与HCV最相似的BVDV株)，可以清楚地看到蒿甲醚的效果。该产品的EC90＝0.16μg/ml。这些实验中发现的毒性浓度在100μg/ml附近。也可以观察到抗BDV的效果(EC90＝0.8μg/ml)。
青蒿素和蒿属粗提取物(KE-4、KE-5和KE-6)都进行了抗肿瘤细胞株L12 10/0、FM3A/0、Molt4/C8和CEM/0的筛选，但没有发现有益结果。
结论
在最初的抗DNA病毒和逆转录病毒的筛选中我们没有发现任何明显的抗病毒活性。在第二次抗BVDV(RNA病毒)的筛选中我们清楚地显示青蒿素、青蒿琥酯、蒿甲醚和双氢青蒿素的强抗病毒活性。这些产品都是内过氧化物，与检测过的蒿属粗提取物相比较，显示明显更好的抗BVDV活性。因为BVDV与HCV有许多相似性，青蒿素、青蒿琥酯、蒿甲醚、双氢青蒿素及其他可能的过氧化物可能具有强的和选择性的抗HCV的抗病毒性质。
尽管本发明按期望已经描述了优选的具体实施方式，但可以理解的是，本发明并不仅仅局限于此，因为对其作出的变化和修饰也属于所附权利要求书中所定义的本发明完整范围内。
1、倍半萜或其药学可接受的盐在制备一种用于治疗由黄病毒科病毒引起的感染的药物中的用途，所述倍半萜具有以下通式：
其中：
Q选自CO、CHOH、CHOCH3、CHOC2H5、和CHOCOCCH2CH2COOH。
2、权利要求1的用途，其中所述感染是丙型肝炎。
3、权利要求1的用途，其中所述感染是牛病毒性腹泻或猪瘟。
The use of endoperoxides for the treatment of infections caused by flaviviridae, including hepatitis c, bovine viral diarrhea and classical swine fever virus
The use of sesquiterpenes and, in particular sesquiterpene lactone endoperoxides, such as artemisinin and analogs thereof, for the treatment of hepatitis C virus infections. Artemisinin, analogs of artemsisnin and some crude Artemisia extracts were tested in vitro against DNA-viruses, retro-viruses and Flavivirida, (an important family of human and animal RNA pathogens). These compounds were also screened for anti-tumor activity. Strong activity of artemisinin was noticed against the bovine viral diarrhea virus (BVDV). As pestiviruses, such as BVDV, share many similarities with hepatitis C virus (HCV), we can conclude that endoperoxides in general and artemisinin more specificly have efficacy as treatments for hepatitis C viral infections.
The application of endoperoxide in the medicine of the infection that the preparation treatment is caused by flaviviridae (comprising hepatitis C virus, bovine viral diarrhea virus and swine fever virus)
Background of invention
The present invention relates generally to the application of the application, particularly sesquiterpene lactones endoperoxide of sesquiterpene lactones class (sesquiterpene lactones) in the infection that treatment is caused by flaviviridae (Flaviviridae) in treatment hepatitis C infection, yellow fever, dengue fever, bovine viral diarrhoea and swine fever (classical swine fever).
Chronic hcv infection is an important public health problem that influences the whole world 1.8 hundred million people (total population 3%), comprises 4 million peoples of the U.S. here, and is a main cause that causes the liver chronic disease.Someone expects that the HCV infection of the U.S. will continue to increase, and will reach present 3 times to the infected in 2010.The infection of hepatitis C virus may cause serious, may be life-threatening chronic hepatopathy in some cases, comprises liver failure and hepatocarcinoma.
In the U.S., chronic hepatopathy is to cause being grown up the tenth dead reason, and causes for 8 every year near 25000 examples or near 1% death toll.Studies show that on the population-based: 40% chronic hepatopathy is that HCV is relevant, causes the annual approximately death of 8000-10000.The medical treatment of the acute and chronic hepatopathy that HCV is relevant and work estimated amount of damage every year will be above 600,000,000 dollars.And the hepatopathy in late period that HCV is relevant is the modal reason of adult orthotopic liver transplantation.Because most HCV the infected's age, the death toll that is caused by the chronic hepatopathy relevant with HCV may significantly increase in the period of the 10-20 in future between 30-49 year, at this time this group the infected will reach the age that the complication of chronic hepatopathy takes place usually.
The propagation of HCV mainly is a large amount of or repeated direct by skin and blood.In the U.S., propagating two kinds of relevant modal contacts with HCV is blood transfusion and shoot up.Therapy for hepatitis C is to change field fast in the clinical practice.The therapeutic alliance of interferon and ribavirin (a kind of nucleic acid analog) has been approved for the treatment first of chronic hepatitis C.The patient of the therapeutic alliance of using ribavirin and interferon be studies confirm that compare with the response rate 15-25% of independent application of interference extract for treating, its persistent response rate has remarkable increase, reaches 40-50%.Most of patients has interferon one property crossed cold like symptoms in early days in treatment, but these symptoms alleviate in continuing treatment.Side effect subsequently has fatigue, bone marrow depression and maincenter mentation (for example, indifferent, cognitive variation, irascible and depressed).Because serious adverse, the dosage of the patient's of 10-40% interferon must reduce, and the patient of 5-15% must be interrupted use.Ribavirin can cause hemolytic anemia, and also is problematic for the unborn anemia of patient, bone marrow depression or renal failure.These patients, should avoid therapeutic alliance, maybe should attempt to correct anemia.The hemolytic anemia that ribavirin causes also may be life-threatening to the patient that ischemic heart disease or cerebrovascular disease are arranged.Ribavirin be cause odd-shaped, female patient in therapeutic process, to avoid gestation.Other treatment comprises corticosteroid, ursodeoxycholic acid (ursodiol) and thymosin, is invalid.The high iron level of liver can reduce the drug effect of interferon.The use in conjunction of de-iron treatment (venotomy or chelating art) and interferon after deliberation, still, also still do not have conclusion ( Www.cdc.gov).So, medical domain press for find with development can be used for treating HCV infect, have simultaneously less or can reduce side effect and compare the new bioactive molecule that better curative effect is arranged with existing treatment.
Summary of the invention
Have been found that sesquiterpene lactones with following general formula:
Have antagonism by flaviviridae, comprise the activity of the infection that hepatitis C virus, bovine viral diarrhea virus and swine fever virus cause.The application has described some representative, at present preferred sesquiterpene lactones classes, and but, those skilled in the art obviously can understand, and other sesquiterpene lactones chemical compounds also are effective in the treatment of the infection that is caused by flaviviridae in the known technology.The pharmaceutically acceptable salt that also comprises this compounds.
The sesquiterpene lactones of preferentially selecting for use is sesquiterpene lactones endoperoxides artemisinin (artemisinin), dihydroarteannuin (dihydroartemisinin), artesunate (artensunate) and Artemether (artemether).
Description of drawings
Fig. 1 is the graphic extension to arteannuin, dihydroarteannuin, artesunate and Artemether chemical constitution.
The specific embodiment
Among the application, we will introduce the antiviral effect of sesquiterpene lactones class, sesquiterpene lactones endoperoxide particularly, for example arteannuin, dihydroarteannuin, artesunate and Artemether are to the specific flaviviridae antivirus action of hepatitis C virus for example.
Preferred sesquiterpenoid of the present invention comprises the chemical compound with following general formula:
Wherein: X 1And X 2Be selected from O, S, Se and NH; Y is selected from O, S, Se and NH; Z is selected from O, NH, S and Se, and Q is selected from CO, CHOH, CHOCH 3, CHOC 2H 5, CHOC 3H 7And CHOCOCCH 2CH 2COOH and drug acceptable salt thereof.
The particularly preferred sesquiterpenoid of the present invention comprises arteannuin, its X 1And X 2Be that O, Y are that O, Z are that O and Q are C=O; Dihydroarteannuin (same arteannuin, but Q is CHOH); Artemether (same arteannuin, but Q is CHOCH 3); Arteether (arteether) (same arteannuin, but Q is COC 2H 5); A kind of propyl group product (same arteannuin, but Q is CHOC 3H 7); And artesunate (artesunate) (same arteannuin, but Q is CHOCOCH 2CH 2COOH).Most preferred sesquiterpenoid of the present invention is a dihydroarteannuin.
Endoperoxide has a dioxy key (peroxo linkage), and (O-O), to be considered to that these products are had the malaria activity be very important to this structure.With-S-S-(disulfide bond), or-Se-Se-(two selenium keys), or-N-O-or-NH-NH-(hydrazine), and the various combination of above-mentioned key can produce new chemical compound, also may have activity.
Flaviviridae is (the Rice CM.1996.Flaviviridae:the viruses and their replication.In:Fields BN of important RNA viruses pathogen section of human and animal, Knipe DM, Howley PM, eds.Fields virology.Philadelphia:Lippincott-Raven Publishers.Pp 931-960).The genus of the flaviviridae of having found at present shows different aspect route of transmission, host range and pathogeny.Typical banzi virus member is yellow fever virus, dengue virus and Pestivirus (pestiviruses), for example bovine viral diarrhea virus (BVDV) and swine fever virus (CSFV).The member of the tool feature that this family is up-to-date is people's a common and proprietary pathogen, i.e. hepatitis C virus (HCV).Banzi virus is to have (+) the polar single strand RNA virus of adopted rna gene group is arranged.Other have (+) has the Viraceae of adopted RNA to comprise Picornaviridae (Picornaviridae), Togaviridae (Togaviridae), Caliciviridae (Caliciviridae) and coronaviridae (Coronaviridae).
The compounds of this invention can be used their original shape or their salt.In certain situation, these chemical compounds can form the stable nontoxic acid or the salt of alkali by enough alkalescence or acidity, and these chemical compounds are used with the form of salt and are fit to.The example of the acceptable salt of pharmacy is can accept the acylate that anionic organic acid forms with the physiology who forms, ascorbic acid acetate for example, benzoate, citrate, etoglutarate, glycerophosphate, malonate, mesylate, succinate and tartrate.Suitable inorganic acid salt also can be synthesized, and comprises heavy carbonate, carbonate, hydrochlorate, nitrate, and sulfate.
The present invention can contain the pharmaceutical composition of described chemical compound and suitable excipient and conventional administration, and said composition antagonism viral infection is useful.According to what treat is interior or outer viral infection, and chemical compound of the present invention and compositions can be through parenteral, part, intravaginal, oral or rectally.
To parenteral, active component or its saline solution can prepare by water, randomly mix with nontoxic surfactant.Also can be at glycerol, liquid macrogol, the mixture of triacetin and mentioned reagent and in oil preparation, preparing.
The spendable dosage of chemical compound can be by determining in its activity of external comparison.Extrapolation method to people's effective dose is known in the art.
This chemical compound can unit dose the conventional administration of form; For example the per unit dosage form comprises active component 0.1-2000mg, is preferably 100-1000mg, most preferably is 100-500mg.Required dose can disposable routinely administration or with suitable interval gradation administration, for example can divide 2,3,4 or more times administration every day.Each sub-doses can also further separately for example be divided into the administration form of some dispersive loose separations; For example repeatedly suck or repeatedly put ocular administration from medicine feeding machine.
To infecting in the body, compositions can be passed through oral or parenteral by the dosage level that free drug calculates, and is approximately 1-30mg/kg (weight of mammal), preferred 1-10mg/kg (weight of mammal).
Must depend on the needs of the individuality of being treated in the accurate scheme of the administration of the chemical compound of this announcement and compositions, the type of treatment also has attending doctor's judgement certainly.Chemical compound of the present invention can be used for having the animal of this treatment demand.Under the situation, be to be used for the people mostly, but domestic animal and house pet also can be expected especially and belonged to range of application of the present invention.
Material and method
Arteannuin is purchased in Aldrich.The plant crude extract of Absinthium (Artemisia absinthum) KE-4, Herba Artemisiae annuae (Artemisia annua) KE-5 and tarragon (Artemisia dracunculus) KE-6 is from U.S. Des Moines, and Kemin Industrial Co., Ltd obtains.In order to prepare KE-4 ,-5 and-6 samples, ground Artemisia absinihium L, sweetness and bitterness Chinese mugwort or the tarragon that 2g is done stirring at room 8 hours in the 200ml hexane, then, suspension filter through the G3 glass filter and low pressure under evaporate.Residual black filtering residue is used for biological screening.Artemether, dihydroarteannuin and artesunate are purchased the Pharma in Dafra.Above-claimed cpd has all carried out the screening of anti-different Causative viruss, for example human immunodeficiency virus (HIV), herpes simplex virus (HSV), chickenpox virus (VV), varicella zoster virus (VZV) and human cytomegalic inclusion disease virus (CMV).To all virus, except CMV, detected EC 50(to HIV in people's cem cell culture inductive cytopathogenicity, HSV and VV at human embryonic fibroblast E 6Inductive cytopathogenicity and VZV inductive plaque in human embryonic lung (HEL) cell culture forms the concentration that reaches the required active compound of 50% inhibition in the SM cell culture).For determination of antiviral activity, anti-CMV IC 50Expression, the human embryonic lung becomes fiber (HEL) cell to cultivate every hole 20PFU viral infection with 96 orifice plates.
After 2 hours, add the culture fluid that 0.1ml contains the gradient dilution of test compounds 37 ℃ of cultivations in the cell of infection.After cell Giemsa dyeing, counted plaque on the 7th day at microscopically.Minimum antiviral concentration reaches 50% required dosage with the plaque formation that suppresses virus induction and represents.
These chemical compounds have also been carried out the screening of anti-flavivirus.Because in fact there are not enough analyzed in vitro to be used for anti-HCV screening, so decision screening anti-bovine viral diarrhea virus (BVDV), because it and hepatitis C virus have a lot of similarity (Frolovl, McBride S and Rice CM.Cis-acting RNA elements required for replication of bovine.Viral diarrheavirus-hepatitis C virus 5 ' non-translated region chimeras.RNA 4,1418-1435 (1998)).Further the effect of the not homophyletic of the anti-BVDV of Artemether is screened.
These chemical compounds have also been carried out the detection of anti-tumor activity, by (Molt4/C8, propagation CEM/0) (detection) is carried out to murine leukemia cell (L1210/0), molluscum contagiosum adenocarcinoma cell (FM3A) and human T lymphocyte.Observed best antiviral activity to CMV and VZV is from KE-6.Next is the antiviral activity of arteannuin to HSV-2 and VV.
Result and discussion
In first screening (seeing Table 1), detected arteannuin and some artemisia crude extract (KE-4, KE-5 and KE-6) antiviral activity to HIV-1, HIV-2, HSV-1, HSV-2, VV, CMV and VZV.
Table 1: the The selection result of anti-HIV-1, HIV-2, HSV-1, HSV-2, VV, CMV and VZV
aThe 50% couple of HIV in human lymphocyte cem cell culture inductive cytopathogenicity, HSV and VV at human embryonic fibroblast E 6Inductive cytopathogenicity and CMV and VZV inductive plaque in human embryonic lung's hel cell culture forms and reaches 50% the required compounds effective concentration of inhibition in the SM cell culture.
Carried out second screening (table 2) in addition, so that detect the activity of anti-bovine viral diarrhea virus in Ren Bovis seu Bubali (MDBK) cell.
Table 2: the The selection result of anti-BVDV in the MDBK cell
Antiviral activity is assessed on the MDBK cell with the Pe515 strain of BVDV.The MTS method is all used in antiviral activity and Cytotoxic detection.
EC 50Be needed concentration when reducing the cytotoxic effect 50% that virus causes.MTC (minimum toxic concentration) is defined as and causes that cellular metabolism reduces＞=20% concentration.
When handling the MDBK cell with Artemether, arteannuin, KE4 and KE5, (100 μ g/ml) still do not reach MTC at maximum concentration.Observe the activity that endoperoxides artemisinin, dihydroarteannuin, Artemether and artesunate have very strong anti-flavivirus BVDV in this test, have low cytotoxicity simultaneously.So these products can be used for the treatment of the infection that is caused by banzi virus.
Carried out the screening (table 3) of anti-different B VDV strain and anti-BDV (border disease virus) with Artemether
Table 3: the The selection result of anti-BVDV of Artemether and BDV
| Artemether | 100 μg/ml | 20 μg/ml | 4 μg/ml | 800 ng/ml | 160 ng/ml | 32 ng/ml | 6.4 ng/ml | 1.28 ng/ml | 0.512 ng/ml | 0.256 ng/ml |
| Non-infection | Toxicity | |||||||||
| BVDVII ncp | Toxicity | ++ | ++ | ++ | +++ | +++ | +++ | +++ | +++ | +++ |
| BVDVI cp | Toxicity | + | + | + | + | ++ | ++ | ++ | ++ | ++ |
| BVDVI ncp | Toxicity | + | + | + | ++ | +++ | +++ | +++ | +++ | +++ |
| BDV | Toxicity | + | + | + | ++ | ++ | ++ | ++ | ++ | ++ |
+++all cells all is male
+++/-50% cell is male
++/-10% cell is male
The BVDV bovine viral diarrhea virus
The BDV border disease virus
The strain of cp cytopathogenic effect
The non-cytopathogenic effect strain of ncp
Anti-especially BVDVIcp (the BVDV strain the most similar to HCV) can be clear that the effect of Artemether.The EC of this product 90=0.16 μ g/ml.The toxic concentration of finding in these experiments is near 100 μ g/ml.Also can observe the effect (EC of anti-BDV 90=0.8 μ g/ml).
Arteannuin and artemisia crude extract (KE-4, KE-5 and KE-6) have all been carried out the screening of antitumor cell strain L12 10/0, FM3A/0, Molt4/C8 and CEM/0, but do not find useful result.
Conclusion
We do not find any tangible antiviral activity in initial resisting DNA virus and retroviral screening.We clearly illustrate the strong antiviral activity of arteannuin, artesunate, Artemether and dihydroarteannuin in the screening of the anti-BVDV second time (RNA viruses).These products all are endoperoxides, compare with the artemisia crude extract that detected, and show obviously better anti-BVDV activity.Because BVDV and HCV have many similaritys, arteannuin, artesunate, Artemether, dihydroarteannuin and other possible peroxide may have strong and the antiviral properties of anti-HCV optionally.
Although the present invention has described the preferred specific embodiment by expectation, be understandable that the present invention is not limited only to this, because to its variation made from modify and also to belong in the appended claims in the defined full breadth of the present invention.
1, sesquiterpene or the acceptable salt of its pharmacy purposes in a kind of medicine that is used for the treatment of the infection that causes by flaviviridae of preparation, described sesquiterpene has following general formula:
Wherein:
Q is selected from CO, CHOH, CHOCH 3, CHOC 2H 5, and CHOCOCCH 2CH 2COOH.
2, the purposes of claim 1, wherein said infection is a hepatitis C.
3, the purposes of claim 1, wherein said infection are bovine viral diarrhoea or swine fever.
0 Comments
Recommended Comments
There are no comments to display.