青蒿素及其衍生物抗疟机制研究概况

WHO的疟疾报告,2017年,估计在87个国家中存在2.19亿疟疾病例;死亡人数估计为43.5万例。每2分钟就有1名儿童死于疟疾。中国自2016年8月以来,未发生1例本土疟疾病例。青蒿素类药物对全球疟疾的控制起到了重要作用,现有的最佳治疗方法,特别是恶性疟治疗方法,仍是以青蒿素为基础的联合疗法。青蒿素全新的化学结构,改写了只有含N杂环的生物碱成分抗疟的历史。但青蒿素类药物的抗疟机制目前仍是久而未决的问题,大湄公河次区域的一些地区也出现了青蒿素耐药性现象,WHO因此发起了"紧急应对青蒿素耐药性"行动。深入探讨青蒿素类药物抗疟机制,不仅可以加深对药物的认识,而且对于指导合理用药、提高临床疗效、防止抗药株的产生、发现新的青蒿素抗疟组合以及拓宽其应用范围都有重要意义。青蒿素抗疟机理解释的难点可能在于青蒿素是原药(本身活性不强),激活和靶标都不确定。目前有关青蒿素类药物抗疟机制主要有以下假说:碳自由基假说、血红素靶标假说、肌内质网Ca~(2+)-ATP酶(钙泵)假说、线粒体靶标假说和血红素激活的多靶标假说。另外也有研究证明疟原虫的膜系结构、血红素聚合产物、一些特定蛋白、诱导疟原虫环状体休眠/沉默、自噬相关蛋白18(ATG18)都可能是青蒿素类药物的作用靶标。但这些假说尚不能单独、完全解释青蒿素的抗疟机制。由于疟原虫的主要致病阶段是红细胞内期的裂体增殖期。下一步的研究工作,除进一步证实上述假说外,可以根据青蒿素选择性杀灭处于红内期的疟原虫、针对疟原虫的膜系结构(表膜-线粒体功能)、疟原虫顶质体内的"蓝藻型"铁代谢通路、染虫红细胞表面膜蛋白、染虫红细胞表面膜离子通道和染虫红细胞改生物力学特征等方面开展研究工作。     

青蒿素的研究

1971年我国再次发现青蒿有抗疟作用，并证实乙醚提取物对数种疟原虫有活性，包括啮齿动物的伯氏疟原虫（P．Bershei）。青蒿的许多化学成分早已被证实，但1971年我国科学家从这植物中又分离出7种倍半能化合物，其中一种是以前未阐明的晶状物，这一物质已命名为青蒿素，主要有抗疟作用。青蒿素在体外对fowleria纳氏阿米巴原虫、日本血吸虫、曼氏血吸虫与华技军吸虫均有活性。本文主要介绍其抗疟作用。1早期药物的发现1971年再次发现青蒿素后不久，经动物试验证实，青蒿素对啮齿动物、鸟与猿猴等疟疾模型中的无性生殖期疟原虫有活性，此种抗…     

新的每日一次疟疾治疗方法开始Ⅲ期试验

一项针对每天一次口服治疗疟疾的药物Pyramax（pyronaridine artesunate，pyronaridine青蒿琥酯）（Ⅰ）的关键Ⅲ期试验已经开始，据报道该药相对于传统的基于青蒿素的联合治疗来说具有更长的保存期。（Ⅰ）被开发用于治疗不复杂的恶性疟原虫疟疾和间日疟原虫疟疾，每天服药一次，共用3天。新的试验目标是确认该药物针对全年龄段，     

云南南部一株恶性疟原虫(P.falciparm)体外连续培养成功

Trager等1979年创建的蜡烛缸法,成功地进行了恶性疟原虫红内期体外连续培养,推动了疟疾免疫的的研究工作。国内77年以来一直延用上海生物制品研究所由海南引种培养成功的Fcc-1和Fcc-2两种恶性疟原虫。我省近年来抗氯喹恶性疟原虫株分布日益扩散,严重地阻碍了灭疟工作的进展。     

新过氧化物的抗疟疾作用

新过氧化物的抗疟疾作用美国ｊｏｈｎｓＨｏｐｋｉｎｓ大学研究开发出一系列结构与青蒿素相似的双球内过氧化物（ｅｎｄｏｏｅｒｏｘｉｄｅ），它与药物前体（ｐｒｏｄｒｕｇ）的作用类似，并被疟原虫血液中的铁活化，抗疟作用相当于青蒿素的１５％。研究人员正设法通过增...     

体外微量测定抗疟药药效的方法

抗疟药常用测定方法一般是在实验动物体内进行,此法费时长,消耗也大,更值得注意的是动物疟原虫对某些药物的反应与人疟原虫不尽相同。虽然近年来国外已建立起人疟猴模型,但目前在我国用此模型进行试验尚无条件。Trager等体外培养人恶性疟原虫成功后,为体外筛选抗疟药提供了可能性。由于体外培养时可变因素多,影响此方法的准确性与重复性。为此本实验试以半数效量法测算抑制疟原虫繁殖的用药量,以测定药物的抗疟效力。经多次重复试验误差范围不大,为体外测定抗疟药提供了一种较方便的方法。     

若干药物对体外培养恶性疟原虫及鼠体内约氏疟原虫抑制作用的比较

用体外培养法观察药物对恶性疟原虫的抑制作用及约氏疟原虫感染小鼠4d抑制试验法,对24种药物进行了比较,结果表明大部分药物在两种方法中表现出相仿的效果,但有个别如羟基脲及洁霉素体外及小鼠试验的结果不尽相符。作者对宜用何种方法作为抗疟药物初筛试验问题作了讨论。     

训化中华按蚊,大劣按蚊吸食恶性疟原虫体外培养液的观察

训化中华按蚊、大劣按蚊吸食恶性疟原虫体外培养液的观察云南省疟疾防治研究所李宗惠关键词恶性疟原虫，体外培养人和按蚊是恶性疟原虫生活史中的两个宿主，随着Ｔｒａｇｅ及Ｊｏｎｓｅｎ体外连续培养恶性疟原虫接种在按蚊上，让其在蚊体内经有性繁殖发育成于孢子，则为研...     

体外培养疟原虫的讨论总结

世界卫生组织与美国国际发展署于1977年3月联合召开过一次关于疟原虫的生物学及体外培养的讨论会,随后发表了专辑。其中有一篇关于红内期疟原虫体外培养讨论的总结,本文系部分内容的摘译。     

抗生育剂棉酚的体外抗疟活性

棉酚正作为男性避孕药广泛地进行研究。晚近研究又显示其抗病毒和抗锥体虫作用。棉酚抗生育的特性被认为是由于抑制精子的乳酸脱氢酶X 活性的结果。疟原虫和精子细胞一样,主要依靠无氧糖酵解供能。因而作者观察了棉酚对人的恶性疟原虫的生长和对寄生虫的乳酸脱氢酶活性的作用。恶性疟原虫(P.falciparum;FCB-1株)是用蜡烛缸体外培养法于人红细胞内培养。培养皿含2.5%     

Simaomicin alpha: effects on the cell cycle of synchronized, cultured Plasmodium falciparum

Simaomicin alpha shows potent antimalarial activity in vitro and is known to be a cell-cycle effector. As erythrocytic schizogony of Plasmodium correlates with cell cycle events, we investigated the effect of simaomicin alpha on stage development of the malaria parasite Plasmodium falciparum. Simaomicin alpha interferes with normal parasite development in a time and concentration dependent manner. Parasites exposed to 2.5 nM simaomicin alpha at the ring stage or trophozoite stage showed disrupted development and immature schizont-like and segmenter-like forms were observed. However, schizont stage parasites were not affected by 2.5 nM simaomicin alpha. It is unclear whether mitosis involved in sequential parasite development occurred when parasites were exposed to simaomicin alpha at the ring or trophozoite stage. At a concentration of 5.0 nM, simaomicin alpha inhibited merozoite-trophozoite development. This concentration curtails p-LDH activity at all parasite stages, although its impact on the schizont stage is delayed for 24 hours.     

Antimalarial effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal by the inhibition of Plasmodium falciparum calpain

The biological understanding of malaria parasites has increased considerably over the past two decades with the discovery of many potential targets for the development of new antimalarial drugs. Calpain, a cysteine protease of Plasmodium falciparum, is believed to be a central mediator essential for parasitic activity. However, the utility of calpain as a potential anti-malarial target in P. falciparum has not been fully determined. In the present study, we determined the effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal (ALLN)-treatment on the expression of calpain in erythrocytic stages of P. falciparum and its usefulness as an antimalarial chemotherapeutic agent. ALLN was shown to have low toxicity to HeLa cells but high toxicity to malaria. ALLN inhibited the expression of calpain in ring, trophozoite and schizont stages when treated for 48 h. Also, after 48 h, samples were characterized by 6.15% and 0% parasitemia without ALLN treatment and with ALLN treatment, respectively. Brightfield and confocal microscopy revealed that ALLN treatment affects merozoite maturation. As ALLN concentration increased from 1 μM to 100 μM, ring stage parasites did not mature into the schizont stage. When ALLN treatment was continued for 48 h, it also significantly inhibited the maturation of ring-stage parasites into trophozoite or schizont stages and survival of malarial parasites. Taken together, these findings suggest that ALLN inhibit the maturation and survival of P. falciparum and calpain expression, and thus has potential utility as an antimalarial chemotherapeutic agent. These two authors made equal contribution to this work.     

Stilbene-chalcone hybrids: design, synthesis, and evaluation as a new class of antimalarial scaffolds that trigger cell death through stage specific apoptosis

Novel stilbene-chalcone (S-C) hybrids were synthesized via a sequential Claisen-Schmidt-Knoevenagel-Heck approach and evaluated for antiplasmodial activity in in vitro red cell culture using SYBR Green I assay. The most potent hybrid (11) showed IC(50) of 2.2, 1.4, and 6.4 μM against 3D7 (chloroquine sensitive), Indo, and Dd2 (chloroquine resistant) strains of Plasmodium falciparum, respectively. Interestingly, the respective individual stilbene (IC(50) > 100 μM), chalcone (IC(50) = 11.5 μM), or an equimolar mixture of stilbene and chalcone (IC(50) = 32.5 μM) were less potent than 11. Studies done using specific stage enriched cultures and parasite in continuous culture indicate that 11 and 18 spare the schizont but block the progression of the parasite life cycle at the ring or the trophozoite stages. Further, 11 and 18 caused chromatin condensation, DNA fragmentation, and loss of mitochondrial membrane potential in Plasmodium falciparum, thereby suggesting their ability to cause apoptosis in malaria parasite.     

Interaction of berbamine and chloroquine or artemisinin against chloroquine-sensitive and -resistant plasmodium falciparum in vitro

Antimalarial activity of berbamine (BB), alone or in combination with chloroquine (CQ) or artemisinin (qinghaosu, QHS), was studied using CQ-sensitive and -resistant strains of Plasmodium falciparum in vitro. BB was found to have antimalarial effects with IC₅₀ values of 603 and 359 nM, respectively, for the drug-sensitive and -resistant strains of P. falciparum, indicating that BB exhibited some selective antimalarial activity against the drug-resistant parasite. Its antimalarial efficacy and selectivity appeared to be less than that of tetrandrine (TT), however, when BB was combined with CQ, an antagonistic interaction was found against the CQ-sensitive parasite, while a potentiating antimalarial action was observed with the CQ-resistant parasite. When BB was tested in combination with QHS, a complex interaction was found—one was additive for the CQ-sensitive parasite and the other was potentiating for the CQ-resistant parasite. The above data suggested that the BB combination with either CQ or QHS is a promising candidate for an antimalarial remedy to treat at least CQ-resistant falciparum parasites which cause malaria. © 1993 wiley-Liss, Inc.     

Semi-mechanistic pharmacokinetic/pharmacodynamic modelling of the antimalarial effect of artemisinin

PURPOSE: To characterize artemisinin pharmacokinetics (PK) and its antimalarial activity in vivo. METHODS: Artemisinin salivary concentration and parasite count data were obtained from Vietnamese malaria patients receiving two different dosage regimens. PK data were analysed using a previously developed semiphysiological model incorporating autoinduction of eliminating enzymes. A pharmacodynamic (PD) model reflecting different stages of the parasite life-cycle was developed and fitted to the data. The model included visible and invisible compartments as well as sensitive, insensitive, and injured parasite stages. Salivary artemisinin concentrations functioned as the driving force for the observed decrease in the number of parasites. RESULTS: Large interindividual variability was observed in both PK and PD data. The PK model described reasonably well the observed decrease in salivary concentrations after repeated drug administration. The preinduction hepatic extraction ratio of artemisinin was estimated to be 0.87 with a volume of distribution of 27 L. Artemisinin half-life averaged 0.7 h. Incorporation of a saturable hepatic elimination affecting the first-pass extraction as well as a higher intrinsic clearance in female patients resulted in the best fit of the model to the data. The PD model described the decrease in the number of parasites during the course of treatment well. The longest mean transit time of parasites from sensitive, visible to invisible to insensitive visible stages was found to be 34.5 h through one life-cycle. The half-life of injured parasites was 2.7 h. CONCLUSIONS: The proposed semimechanistic PK/PD model successfully described the time course of both salivary artemisinin concentrations after repeated dosing and the number of parasites in patients treated with the drug.     

Dual Targeting of Function–Structure for Effective Killing of Pathogenic Free-Living Amoebae

Here we discuss the potential value of targeting both the hardy structure of the cyst state of the parasite, in addition to the active trophozoite form, to provide target-directed inhibition as a viable drug strategy in the effective eradication of parasites.     

伯氏疟原虫对青蒿素抗药性的研究

仿Peters剂量递增法用伯氏疟原虫ANKA株及N株对QHS进行了抗药性的研究。经14个月的培育至第58代,QHS im注射“4日抑制性实验”的ED_(50)在RQ/ANKA系及RQ/N系分别为其亲代系的53.4及54.6倍,但经蚊传未获成功。在第40代(I_(50)=25)时,其50%的治愈剂量为其亲代系的5.4倍。停药传代其抗性会逐渐消失。该虫系对青蒿酯钠及蒿甲醚有明显的交叉抗性,其ED_(50)分别为其亲代系的13.1及11.7倍,对伯喹的抗性为2.9倍,对氯喹未见明显交叉抗性。     

Synthesis of thiazolyl hydrazonothiazolamines and 1,3,4‐thiadiazinyl hydrazonothiazolamines as a class of antimalarial agents

Novel thiazolyl hydrazonothiazolamines and 1,3,4‐thiadiazinyl hydrazonothiazolamines were synthesized by a facile one‐pot multicomponent approach by the reaction of 2‐amino‐4‐methyl‐5‐acetylthiazole, thiosemicarbazide or thiocarbohydrazide and phenacyl bromides or 3‐(2‐bromoacetyl)‐2H‐chromen‐2‐ones in acetic acid with good to excellent yields. These new compounds were screened in vitro for their antimalarial activity; among them, four compounds, 4h, 4i, 4k, 4l , showed moderate activity with half‐maximal inhibitory concentration (IC₅₀) values of 3.2, 2.7, 2.7, and 2.8 and 3.2, 3.2, 3.1, and 3.5 μM against chloroquine‐sensitive and ‐resistant strains of Plasmodium falciparum, respectively. Compound 4l inhibited the ring stage growth of P. falciparum 3D7 at an IC₉₀ concentration of 12.5 µM in a stage‐specific assay method, where the culture is incubated with specific stages of P. falciparum for 12 hr, and no activity was found against the trophozoite and schizont stages, confirming that 4l may have potent action against the ring stage of P. falciparum.     

Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes: growth inhibition in vitro

Blocking spermidine and spermine synthesis in Plasmodium falciparum-infected erythrocytes with irreversible inhibitors of S-adenosylmethionine decarboxylase (AdoMet DC; EC 4.1.1.50), prevented the growth of the parasite in vitro. The most potent of these compounds, MDL 73811, inhibited growth of chloroquine-sensitive and -resistant strains of P. falciparum equally, with an IC50 of 2-3 microM. Other structurally related compounds also inhibited parasite proliferation, but to a lesser degree, determined apparently by their potency for inhibition of AdoMet DC. The growth inhibition by MDL 73811 could be alleviated by incubating infected erythrocytes with spermidine and spermine, but not putrescine. Parasites treated with the drug were arrested at the trophozoite stage of the erythrocytic cycle and had putrescine levels which were elevated by about 3- to 4-fold. Treatment of crude extracts of purified parasites with 1 microM MDL 73811 inhibited AdoMet DC activity by greater than 90%. These biochemical changes in P. falciparum-infected cells were consistent with AdoMet DC inhibition being the primary effect of MDL 73811 treatment.