中国青蒿素生产企业的困境与对策分析

目的:探讨解决青蒿素生产企业所面临的困境的方法。方法:运用市场结构理论深入分析了中国青蒿素生产企业面临的困境及形成的原因,并给出了具体的政策建议。结果与结论:中国青蒿素生产企业应该从整合青蒿素上游资源,组建行业协会,增强研发等方面来解决所面对的困境。     

我国青蒿素类抗疟复方制剂进入越南市场浅析

目的:为我国青蒿素类抗疟复方制剂进入越南市场提供理论分析依据。方法:通过文献调研、资料归纳、综合分析等手段,介绍越南的青蒿素类抗疟药市场概况,并分析进入越南市场的必要性、存在问题和制约因素。结果与结论:我国青蒿素类抗疟复方制剂生产企业要想进入越南市场并获得成功,必须选择合适的经营模式,树立良好的药品品牌,引导销售,并加强与医患双方的沟通,以及开展国际合作等。     

多孔淀粉-环糊精基青蒿素微球在大鼠体内的生物利用度和组织分布研究

目的:探讨一种多孔淀粉-环糊精基青蒿素微球(AHPS)在大鼠体内的生物利用度和组织分布规律.方法:通过对大鼠分别灌胃给药青蒿素微球后,测定青蒿素的血液中的药物浓度,考察在大鼠体内青蒿素微球不同的吸收和代谢程度;同时,经过灌胃AHPS后,观察在大鼠心、肝、脾、肺、肾、脑等6个器官中青蒿素的分布,探讨青蒿素微球吸收入血后青蒿素在大鼠体内的分布情况.结果:与青蒿素以及青蒿素哌喹片相比,青蒿素微球在大鼠体内的血药达峰浓度有了很大程度的提升,生物利用度为青蒿素的4.46倍、青蒿素哌喹片的3.08倍;并且AHPS、青蒿素和青蒿素哌喹片在心脏和肝脏中血药浓度最高,其中AHPS在不同时间各个组织中的含量均比青蒿素和青蒿素哌喹片高.结论:AHPS相比于青蒿素和青蒿素哌喹片,在体内的吸收效果更佳,在各器官中的含量明显增加.此微球为提高青蒿素等难溶于水的药物的生物利用度和体内分布提供理论依据.     

黄花蒿发根的液体培养及青蒿素合成的动态特征研究

目的:探讨黄花蒿发根生长及青蒿素生物合成的动态特征,建立高效、稳定的黄花蒿发根液体培养体系。方法:测定不同培养基以及MS培养基中不同营养元素对黄花蒿发根生物量和青蒿素含量的影响。结果:筛选出优化的黄花蒿发根液体培养基,获得拟合的黄花蒿发根生长和青蒿素合成的Logistic方程。结论:在优化的MS培养基中黄花蒿发根生长迅速且能稳定合成青蒿素,为工业化大规模生产青蒿素提供了可能。     

青蒿中青蒿酸、青蒿素及去氧青蒿素同时提取及检测

目的:建立青蒿中青蒿酸、青蒿素及去氧青蒿素的提取及其含量测定方法。方法:石油醚回流提取3种倍半萜,GC-MS联用测定其含量。结果:3种倍半萜在各自的检测限内线性关系良好(r≥0.999),该方法的稳定性(RSD<5.0%)、重复性(RSD<5.0%)及精密度(RSD<5.0%)良好。结论:该方法简单、精确可靠,尤其适宜监控不同采收期青蒿酸、青蒿素及去氧青蒿素的含量。     

超声萃取-紫外分光光度法测定不同产地青蒿中的青蒿素

目的:对比研究了索氏法和超声法提取、测定青蒿中的青蒿素。方法:选择了索氏提取法、超声萃取法提取植物青蒿中有效成分青蒿素的优化条件,紫外分光光度法直接测定不同产地青蒿中青蒿素的含量。结果:索氏提取法优化条件为时间4h,液固比200:1,提取次数为2次;超声波提取法优化条件为功率70W,时间5min,温度50℃,液固比150:1。平均回收率达98.6%,RSD=2.7%。采用超声法测定多个不同产地青蒿中青蒿素的含量优于索氏法。结论:超声萃取法具有简便、迅速、灵敏度高等特点。     

联合应用青蒿素类化合物抗肿瘤研究进展

目的:探讨联合应用青蒿素类化合物抗肿瘤的意义。方法:通过PubMed检索系统搜索近年来国内、外发表的有关青蒿素类化合物联合其他化合物抗肿瘤的文献,对其进行归纳、整理。结果:青蒿素类化合物与多种化合物,如氨乙酸硫酸亚铁、顺铂、卡铂、吉西他滨、环磷酰胺等联合使用,均具有一定的抗肿瘤增效作用。结论:青蒿素类化合物自身毒性小,与多种化合物联合应用具有增效作用,因此研究联合应用青蒿素类化合物抗肿瘤的课题值得关注。     

青蒿素与过氧化物酶作用的荧光研究

目的:研究青蒿素与过氧化物酶间的作用关系,建立青蒿素荧光检测方法。方法:以吡罗红B为指示剂,用荧光降低法研究青蒿素与辣根过氧化物酶的相互作用。结果:青蒿素与辣根过氧化物酶之间的催化反应米氏常数Km为2.72×10-5mol/L,最大反应速度vmax为1.74×10-4mol/(L·s),催化常数Kcat为24.27s-1。反应作用位点分别是青蒿素内过氧基团与酶中心离子Fe。该反应可用于对黄花蒿药材中青蒿素含量的测定,测定工作曲线为c=0.1684?F-0.8143(cartemisinin单位为1.0×10-7mol/L),相关系数r=0.9965,3σ检出限为3.58×10-8mol/L。结论:青蒿素与辣根过氧化物酶之间的作用具有酶与底物反应的特征,该特征反应可用于对黄花蒿药材中青蒿素含量的测定。     

我国黄花蒿中青蒿素含量的气候适宜性等级划分

针对黄花蒿种植中适生地选择的迫切要求,本文通过实地调查和查阅文献,获得全国各地青蒿素的含量数据。应用统计分析方法研究青蒿素含量与气候因子和地理分布之间的关系,应用ArcGIS软件的空间计算方法进行青蒿素含量的气候适宜性等级划分。结果显示:①青蒿素含量在我国各地差异较大,青蒿素含量纬向变异明显,北部高纬度地区青蒿素含量较低,南部青蒿素含量较高。北纬34度以南,东经(100～120°E)之间地区适宜黄花蒿的生长,青蒿素含量相对较高。北纬34度以北高纬度地区不适宜黄花蒿的生长,而且青蒿素含量低于0.2%。②在我国,亚热带湿润气候区最适宜黄花蒿的生长,而且青蒿素含量平均值大于0.5%。③温度、日照时数和降雨量是影响青蒿素含量高低的主要气候因子。最适宜青蒿素积累的气候条件为:温度(13.9～22℃)、日照时数(853～1 507 h)、降雨量(814～1 518 mm)。最适宜黄花蒿生长的气候条件为:温度(13～29℃)、降雨量(600～1 300 mm)。④广西西北部,四川、贵州、云南东部,重庆南部和湖南西部的气候条件最适宜黄花蒿的人工种植;湖北、安徽和江苏的南部地区也有适宜黄花蒿人工种植的气候条件。     

RP—HPLC法测定O／W型青蒿素脂肪乳剂中青蒿素的含量

目的:建立柱前衍生化 HPLC 法测定 O/W 型青蒿素脂肪乳剂中青蒿素含量的方法。方法:采用青蒿素的柱前碱性衍生反应,Agilent HC—C_(18)(250 mm×4.6 mm,5μm)色谱柱,流动相为甲醇:0.05 moL·L~(-1)醋酸钠-醋酸缓冲液(pH5.8)=66:34;流速:0.5 mL·min~(-1);柱温:25℃;检测波长:259.8 nm。结果:青蒿素的柱前衍生化反应彻底可靠,该方法在检测波长259.8 nm,试样浓度0.3～10μg·mL~(-1)范围内,与检测峰峰面积呈良好的线性关系(r=0.999),平均回收率为96.1%,RSD=1.1%。结论:本方法简便、精密,回收率与精密度高,重现性好,可用于青蒿素脂肪乳剂中青蒿素含量的测定。     

Multiple dose study of interactions between artesunate and artemisinin in healthy volunteers

AIMS: To investigate whether coadministration of the antimalarials artesunate and artemisinin alters the clearance of either drug. METHODS: Ten healthy Vietnamese males (Group AS) were randomized to receive a single dose of 100 mg oral artesunate (pro-drug of dihydroartemisinin) on day -5 and then once daily for 5 consecutive days (days 1-5). Oral artemisinin (500 mg) was coadministered on days 1 and 5. Another 10 subjects (Group AM) were given 500 mg oral artemisinin on day -5 and then further doses on days 1-5. Artesunate 100 mg was given on days 1 and 5. Artemisinin and dihydroartemisinin plasma concentrations on days -5, 1 and 5 were quantified by h.p.l.c. with on-line postcolumn derivatization and u.v. detection. RESULTS: In Group AS, dihydroartemisinin oral clearance values (mean (95% CI)) were similar on day 1 (32 (22, 47)) l h(-1) and day 5 (38 (28, 51)) l h(-1) of daily artesunate administration but these mean values were approximately three fold higher compared with day -5 after a single dose (95 (56, 159)). In this group, artemisinin oral clearance increased from 196 (165, 232) l h(-1) on day 1-315 (241, 410) l h(-1) on day 5. In Group AM, dihydroartemisinin oral clearance on day 1 was 39 (34, 46) l h(-1) and increased 1.6 fold to 64 (48, 85) l h(-1) on day 5. In this group, artemisinin oral clearance increased sequentially (1.5 and 4.7 fold, respectively) from 207 (151, 285) l h(-1) on day -5-308 (257, 368) l h(-1) on day 1 and to 981 (678, 1420) l h(-1) on day 5. The increase in artemisinin oral clearance between days -5 and 1 (in the absence of artesunate) was similar to that between days 1 and 5 in Group AS subjects who took daily artesunate. Dihydroartemisinin was not a significant metabolite of artemisinin. CONCLUSIONS: Artesunate (dihydroartemisinin) did not alter the elimination of artemisinin. However, dihydroartemisinin elimination was inhibited by artemisinin. Artemisinin induced its own elimination even 5 days after a single oral dose. There was no evidence for the formation of dihydroartemisinin from artemisinin.     

A semiphysiological pharmacokinetic model for artemisinin in healthy subjects incorporating autoinduction of metabolism and saturable first-pass hepatic extraction

AIMS: Previous studies have shown that the antimalarial drug artemisinin is a potent inducer of its own metabolism in both patients and healthy subjects. The aim of this study was to characterize the time-dependent pharmacokinetics of artemisinin in healthy subjects. METHODS: Twenty-four healthy males were randomized to receive either a daily single dose of 500 mg oral artemisinin for 5 days, or single oral doses of 100/100/250/250/500 mg on each of the first 5 days. Two subjects from each group were administered a new dose of 500 mg on one of the following days after the beginning of the study: 7, 10, 13, 16, 20, or 24. Artemisinin concentrations in saliva samples collected on days 1, 3, 5, and on the final day were determined by HPLC. Data were analysed using a semiphysiological model incorporating (a) autoinduction of a precursor to the metabolizing enzymes, and (b) a two-compartment pharmacokinetic model with a separate hepatic compartment to mimic the processes of autoinduction and high hepatic extraction. RESULTS: Artemisinin was found to induce its own metabolism with a mean induction time of 1.9 h, whereas the enzyme elimination half-life was estimated to 37.9 h. The hepatic extraction ratio of artemisinin was estimated to be 0.93, increasing to about 0.99 after autoinduction of metabolism. The model indicated that autoinduction mainly affected bioavailability, but not systemic clearance. Non-linear increases in AUC with dose were explained by saturable hepatic elimination affecting the first-pass extraction. CONCLUSION: Artemisinin produces a rapid onset of enzyme induction, resulting in a decrease in its own bioavailability over time. The proposed model successfully described the time-course of the onset and normalization of the autoinduction of metabolism in healthy subjects receiving two different dosage regimens of the compound.     

硅胶柱层析纯化青蒿素

目的确定硅胶柱层析对超临界CO2萃取所得青蒿素粗品纯化的工艺条件。方法采用薄层层析-硅胶柱层析法考察不同展开剂的层析效果,最优洗脱剂为正己烷-乙醚(80∶20);以青蒿素回收率和平均含量为评价指标,考察了流速、进样量与层析柱再生条件对层析分离的影响。结果硅胶柱层析纯化青蒿素的最佳操作条件为洗脱剂空塔流速0.5cm.min-1,进样量18.9mg.ml-1柱体积,甲醇再生2倍床层体积。青蒿素含量可由原来的15%提高到70%以上,回收率达90%。经过重结晶精制,产品中青蒿素含量超过99.5%。结论硅胶柱层析纯化青蒿素所得产品符合质量要求。     

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.     

青蒿素抗肿瘤作用机制研究

目的:运用基因芯片技术检测青蒿素作用于人红白血病细胞株K562细胞后的基因表达情况,从分子水平探讨青蒿素抑制人红白血病细胞株K562增殖的作用机制。方法:人红白血病细胞株K562细胞经不同浓度青蒿素处理24h,用倒置显微镜和荧光显微镜观察细胞形态学变化;流式细胞仪检测细胞周期变化;提取总RNA,逆转录生成cDNA,同时Cy3标记,标记好的cDNA与基因芯片杂交,用扫描仪检测杂交结果。结果:倒置显微镜下可见细胞出现不同程度的皱缩,核分裂相减少,细胞密度下降,漂浮细胞增多;荧光显微镜下可观察到染色质高度浓缩、边缘化,凝聚成明亮的团块,即凋亡小体;流式细胞仪检测显示:G2期细胞的比例显著增加;扫描信号分析数据,其中10条基因表达下调:cyclinD1、cdk4、cdk2、cdc2、DNA-PK、DNA-TopoI、mcl-1、erk、jnk、VEGF。结论:青蒿素可抑制人红白血病细胞株K562细胞增殖,作用机制与改变细胞周期某些调控物质的基因表达、诱导人红白血病细胞株K562细胞凋亡等有关。     

In vitro evidence for auto-induction of artemisinin metabolism in the rat.

Artemisinin disappearance rate was more rapid in incubations with liver microsomes from rats pre-treated with oral artemisinin (60 mg/kg/day for 5 days) compared with microsomes from control animals. A single pathway Michaelis-Menten saturable elimination model was fitted to the concentration-time data of artemisinin incubations by non-linear regression. Model parameters were obtained after fitting results for each animal separately and by pooling data for pre-treated and control animals. Parameter estimates (% coefficient of variation) from fitting the pooled data was maximum velocities (Vmax) = 1.8 (12) mmole/min/mg protein and Michaelis constants (Km) = 20(22) microM for artemisinin pre-treated and Vmax = 0.85 (35) mmole/min/mg protein and Km = 67(52) microM for control animals indicating a 2-fold increase in Vmax and a 3-fold decrease in Km with microsomes from artemisinin pre-treated animals. Estimates of intrinsic clearance in microsomes from the pre-treated animals were 8-fold higher compared with controls. Thus, artemisinin appears to be a potent auto-inducer of drug metabolism in rats as has also been observed in humans. The present findings suggest caution in the interpretation of repeat-dose rat toxicity studies with artemisinin unless its pharmacokinetics are simultaneously monitored, since during multiple administration, the exposure of the drug will not be constant over time.     

Artemisinin pharmacokinetics in healthy adults after 250, 500 and 1000 mg single oral doses

Eight healthy male, Vietnamese subjects were administered 1×250, 2×250 and 4×250 mg artemisinin capsules in a cross-over design with randomized sequence with a 7-day washout period between administrations. The inter-individual variability in artemisinin pharmacokinetics was large with parameter coefficients of variation (CV) typically between 50–70%. The parameter with the smallest variability was the elimination half-life (CV≈30–40%). Analysis of variance indicated also a large intra-subject variability (CV≤24%) for the dose-normalized area under the plasma concentration–time curve (AUC/dose). The pharmacokinetic results suggested artemisinin to be subject to high pre-systemic extraction. Artemisinin half-life could not predict the extent ofin vivo exposure to the drug, there being no correlation between half-life and oral clearance. Artemisinin oral plasma clearance was about 400 L h⁻¹ exhibiting a slight decrease with dose, although the effect was weak. Thus results from studies using different artemisinin doses may, within the studied dose range, be compared without the complication of disproportionate changes in drug exposure with varying dose levels. Half-lives appeared to increase with dose. An observed period effect in the analysis of variance was tentatively associated with time-dependency in artemisinin pharmacokinetics. There was a high correlation between artemisinin plasma concentrations determined at various time-points after drug administration and the AUCs after the 500 and 1000 mg doses, but less so after the 250 mg dose. This may show a tentative approach to assess the systemic exposure of the patients to artemisinin from the determination of artemisinin plasma concentrations in one or two plasma samples only. Artemisinin was well tolerated with no apparent dose or time dependent effects on blood pressure, heart rate or body temperature. © 1998 John Wiley & Sons, Ltd.     

Population pharmacokinetic and pharmacodynamic modelling of artemisinin and mefloquine enantiomers in patients with falciparum malaria

OBJECTIVES: The aims of this study were to investigate whether artemisinin influences the pharmacokinetics of mefloquine enantiomers or vice versa and to model the antiparasitic effect of these drugs alone and in combination in Plasmodium falciparum malaria patients. METHODS: Forty-two male and female patients were randomised to treatment with either oral artemisinin 500 mg daily for 3 days followed by oral mefloquine 750 mg on day 4, oral artemisinin 500 mg daily for 3 days plus oral mefloquine 750 mg on day 1 or a single 750-mg oral dose of mefloquine. The data was modelled using NONMEM. RESULTS: All patients were successfully treated regardless of treatment. The fastest parasite clearance rates were observed in patients receiving artemisinin together with mefloquine on the first day of treatment. A pharmacodynamic model based on the life cycle of P. falciparum successfully described the efficacy of artemisinin, mefloquine and the combination. The time artemisinin concentration stays above a minimum inhibitory concentration was estimated to 2.97 h (relative standard error 4.7 h). The two mefloquine enantiomers exhibited different pharmacokinetics, with an oral clearance of 3.51 (7.9) l/h and 0.602 (6.9) l/h for RS-mefloquine and SR-mefloquine, respectively. In patients receiving only artemisinin the first 3 days, artemisinin oral clearance was 6.9-fold higher the last day of treatment compared with the first day. There was no difference in the pharmacokinetics of mefloquine enantiomers when mefloquine was given alone, in combination with artemisinin or after a 3-day regimen of artemisinin. There was a tendency towards, although non-significant, higher artemisinin concentrations when artemisinin was given together with mefloquine compared with when given alone. CONCLUSIONS: No significant pharmacokinetic interactions were observed after co-administration of artemisinin and mefloquine. The P. falciparum malaria pharmacodynamic model successfully described the antimalarial effect of artemisinin, mefloquine and a combination of the two drugs.     

Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin

AIMS: The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin. METHODS: Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities. RESULTS: The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree. CONCLUSIONS: Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.