Ultrasound-Mediated Microbubble Destruction Suppresses Melanoma Tumor Growth

Melanoma is one of the most aggressive types of cancer, and its incidence has increased rapidly in the past few decades. In this study, we investigated a novel treatment approach, the use of low-intensity ultrasound (2.3 W/cm² at 1?MHz)-mediated Optison microbubble (MB) destruction (UMMD) to treat melanoma in a flank tumor model. The effect of UMMD was first evaluated in the melanoma cell line B16 F10 (B16) in vitro and then in mice inoculated with B16 cells. MB⁺B16 cells were exposed to US in vitro, resulting in significant cell death proportional to duty cycle (R²?=?0.74): approximately 30%, 50%, 80% and 80% cell death at 10%, 30%, 50% and 100% DC respectively. Direct implantation of tumors with MBs, followed by sonication, resulted in retarded tumor growth and improved survival (p?=?0.0106). Immunohistochemical analyses confirmed the significant changes in expression of the cell proliferation marker Ki67 (p?=?0.037) and a microtubule-associated protein 2 (p?=?0.048) after US?+?MB treatment. These results suggest that UMMD could be used as a possible treatment approach in isolated melanoma and has the potential to translate to clinical trials.     

Ultrasound-Mediated Microbubble Destruction Increases Renal Interstitial Capillary Permeability in Early Diabetic Nephropathy Rats

Diabetic nephropathy (DN) is defined as persistent proteinuria corresponding to a urinary albumin excretion rate >300 μg/mg in the absence of other non-diabetic renal diseases. The aim of this study was to determine if ultrasound (US)-mediated microbubble (MB) destruction could increase renal interstitial capillary permeability in early DN rats. Diabetes was induced with streptozotocin. DN rats presented with mild micro-albuminuria 30 d after onset of diabetes. DN rats (N = 120) were divided into four groups that received Evans blue (EB) followed by: (i) no treatment (control group); (ii) continuous ultrasonic irradiation for 5 min (frequency = 7.00 MHz, mechanical index = 0.9, peak rarefactional pressure = 2.38 MPa: US group); (iii) microbubble injection (0.05 mL/kg: MB group); and (iv) both ultrasound and microbubble injection (US + MB group). Another 8 DN rats were subjected to ultrasound and microbubbles and then injected with EB after 24 h (recovery group). EB content, EB extravasation and E-selectin mRNA and protein expression significantly increased, and interstitial capillary walls became discontinuous in the US + MB group. Neither hemorrhage nor necrosis was observed on renal histology. Urine samples were collected 24 h post-treatment. There was no hematuria, and the urinary albumin excretion rate did not increase after ultrasound-microbubble interaction detected by urinalysis. EB content returned to the control group level after 24 h, as assessed for the recovery group. In conclusion, ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability in DN rats, and should be considered a therapy for enhancing drug and gene delivery to the kidney in the future.     

超声微泡造影剂对心肌组织的生物学效应及介导VEGF基因转染大鼠心肌的实验研究

目的探讨超声微泡造影剂对心肌组织的生物学效应及其介导VEGF基因转染大鼠心肌的有效性. 方法 18只健康雄性Wistar大鼠,取3只采用超声波在鼠胸壁破坏微泡造影剂,观察对心肌组织显微结构的影响.将另15只急性心肌梗死3天后的雄性Wistar大鼠分为3组,每组5只.第一组采用超声破坏微泡造影剂的方式,将pcD2VEGF121基因转染大鼠心肌至造影剂不再显影(约6min);第二组尾静脉输入同等剂量携pcD2VEGF121基因的造影剂;第三组为对照.2周后,取缺血心肌组织行VEGF免疫组织化学染色,观察心肌组织血管内皮生长因子(VEGF)蛋白表达情况.结果超声波破坏微泡造影剂能使心肌组织充血,产生大量空泡,并有部分心肌细胞坏死.采用超声微泡造影剂介导的VEGF基因转染,能明显增强大鼠心肌组织VEGF蛋白的表达.结论超声微泡造影剂能明显增强对组织的空化效应,其介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法.     

Partial Restoration of Left Ventricular Systolic Function by asPLB Gene Transfer Using Ultrasound-Mediated Microbubble Destruction

In vitro and in vivo studies have demonstrated that inhibition of phospholamban (PLB) expression in myocardium can restore left ventricular systolic function in failing heart. Ultrasound mediated microbubble destruction provides a new option for noninvasive gene transfer in heart. In this study, we transfered pAAV-antisense phospholamban (pAAV-asPLB) to the hearts of myocardial infarction (MI) mice, using ultrasound mediated microbubble destruction. Then we estimated the protein levels of PLB, Ser16-PLB and cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA). The left ventricular ejection fraction (LVEF), fraction shortening (FS) and SERCA activity were measured as well. MI mice were generated by ligating the left anterior descending coronary artery. Microbubbles were prepared by sonicated perfluorocarbon gas with dextrose and albumin. A mixture of pAAV-asPLB plasmid and microbubble was injected via tail vein while the heart was simultaneously exposed to ultrasound via transthoracic insonation. Three weeks later, LVEF (48.2 ± 5.18% vs 39.1 ± 5.38%, p < 0.05), FS (19.6 ± 2.59% vs 16.0 ± 2.29%, p < 0.05), SERCA activity (3.00 ± 0.29 vs 2.12 ± 0.30, p < 0.05) and Ser16-PLB protein level (0.8 ± 0.25 vs 0.46 ± 0.18, p < 0.05) were increased while PLB protein level (1.45 ± 0.38 vs 2.05 ± 0.31, p < 0.05) was decreased compared with the MI mice with saline injection. The above parameters in MI mice with only pAAV-asPLB plasmid injection or pAAV-asPLB plasmid combined with ultrasound alone were not significantly improved. pAAV-LacZ was used as a reporter gene to determine the efficiency and localization of transfection. The expression of β-galactosidase was not found in liver, lung and brain, but found only in tubular epithelial cells of kidney and found in heart. These results confirm that asPLB gene transfection can be achieved by ultrasound mediated microbubble destruction with organ specificity. The effective transfection can partly restore heart function in MI mice. (E-mail: s0hu0001@hotmail.com)     

超声微泡造影剂对心肌组织的生物学效应及介导VEGF基因转染大鼠心肌的实验研究

目的 探讨超声微泡造影剂对心肌组织的生物学效应及其介导VEGF基因转染大鼠心肌的有效性。方法 18只健康雄性Wistar大鼠，取3只采用超声波在鼠胸壁破坏微泡造影剂，观察对心肌组织显微结构的影响。将另15只急性心肌梗死3天后的雄性Wistar大鼠分为3组，每组5只。第一组采用超声破坏微泡造影剂的方式，将pcDzVEGFm基因转染大鼠心肌至造影剂不再显影(约6min)；第二组尾静脉输入同等剂量携pcD。VEGF。基因的造影剂；第三组为对照。2周后，取缺血心肌组织行VEGF免疫组织化学染色，观察心肌组织血管内皮生长因子(VEGF)蛋白表达情况。结果超声波破坏微泡造影剂能使心肌组织充血，产生大量空泡，并有部分心肌细胞坏死。采用超声微泡造影剂介导的VEGF基因转染，能明显增强大鼠心肌组织VEGF蛋白的表达。结论 超声微泡造影剂能明显增强对组织的空化效应，其介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法。     

超声波介导微泡破裂促EGFP基因转染大鼠脑组织的实验研究

目的 探讨超声波介导微泡造影剂破裂促外源基因在中枢神经系统转染的可行性.方法 15只大鼠分3组,1组经股静脉输入含绿色荧光蛋白质粒(pEGFP)的造影剂0.8 ml,立即用超声波照射大鼠颅骨3 min;第2组输入相同的造影剂0.8 ml,不采用超声波照射;第3组输入不含造影剂的pEGFP 0.2 ml,立即超声波照射3 min.48 h后处死大鼠,荧光显微镜下观察绿色荧光蛋白表达.结果 只在股静脉输入含pEGFP的造影剂,并经超声波照射的大鼠微血管壁上观察到绿色荧光蛋白表达.结论 以微泡造影剂为基因载体,通过超声波靶向破坏微泡,有可能在脑血管内皮细胞中获得基因转染.     

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 10⁸ MBs/mL AP-Cav (8 μM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 μM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.     

脉冲高强度聚焦超声辐照离体组织所致组织摧毁术的实验研究

目的 研究高声功率,低占空比的脉冲高强度聚焦超声(pulsed high intensity focused ultrasound,pHIFU)辐照离体牛肝组织所致组织摧毁术的可行性及机制.方法 使用500 W声功率、4 Hz脉冲重复频率、1％～4％占空比的pHIFU以20 mm深度辐照离体牛肝组织60、30、20 s和15 s.辐照中,使用热电偶和被动空化检测(passive cavitation detection,PCD)系统分别对焦域处的温度和空化信号进行检测.辐照结束后,切开牛肝,观察损伤形态,进行HE染色.结果 pHIFU辐照离体牛肝组织后均有损伤形成,占空比为1％～3％的pHIFU辐照后损伤为洞状;占空比为4％的pHIFU辐照后损伤为凝固性坏死.镜下观察,洞状损伤边缘处呈絮状结构,细胞结构完全破坏,胞浆均质状,细胞核消失.占空比为1％～4％的pHIFU辐照中焦域处的最高温度分别为(41.19±1.42)℃、(45.73±1.92)℃、(53.07±2.09)℃和(64.13±2.56)℃.PCD系统所测信号的频谱中均可见明显的宽带噪声.结论 高声功率,低占空比的pHIFU辐照离体牛肝组织可致组织摧毁术,且空化参与了组织摧毁术的实施.     

Ex Vivo Imaging of Ultrasound-Stimulated Metabolic Activity in Rat Pancreatic Slices

Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 and 633 nm to image lipodamine dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence, respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm² at both 800 kHz and 1 MHz. Additionally, 800 kHz at 1 W/cm² was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.     

白血病肿瘤相关抗原特异性细胞毒T细胞的体外扩增及杀伤功能的实验研究

目的:探索体外扩增白血病肿瘤相关抗原特异性细胞毒T细胞(tumor-associated antigen-specific cytotoxic T lymphocytes,TAA-CTL)的可行性,并验证其特异性杀伤作用。方法:采用密度梯度离心法分离出健康志愿者外周血单个核细胞,诱导培养树突状细胞(dendritic cells,DC)并负载白血病相关抗原WT1、PRAME、NY-ESO-1混合多肽,然后与自体T淋巴细胞共培育,扩增出TAA-CTL。用流式细胞术检测TAA-CTL表型及多种细胞因子的分泌率,细胞毒实验检测TAA-CTL对负载肿瘤相关抗原的自体靶细胞的杀伤力。结果:1体外诱导培养的TAACTL扩增倍数为3.81±1.61;流式细胞术检测其中CD3+细胞平均为(97.22±0.71)%,CD3+CD4+占(41.47±27.08)%,CD3+CD8+占(56.40±11.15)%,CD3-CD56+占(0.50±0.31)%,CD19+仅占(0.14±0.20)%,与对照组细胞表型分析比较差异无统计学意义(P0.05)。2流式细胞术检测经抗原刺激后TAA-CTL分泌的胞内细胞因子,CD8+TAA-CTL分泌的IFN-γ和TNF-α水平分别为(27.67±2.21)%和(34.2±0.71)%,CD4+TAA-CTL分泌的IFN-γ和TNF-α分别为(21.6±2.55)%和(9.97±3.44)%;对照组CD8+CTL分泌的IFN-γ和TNF-α水平分别为(1.36±0.04)%和(5.58±0.03)%,CD4+CTL分泌的IFN-γ和TNF-α分别为(0.91±0.06)%和(1.60±0.07)%,均明显低于TAA-CTL组,其差异有统计学意义(P0.01)。TAA-CTL在效靶比为5∶1、10∶1、20∶1和40∶1时对负载TAA的自体靶细胞的杀伤率分别为(26.85±5.25)%、(60.55±2.45)%、(67.4±3.60)%和(77.00±1.00)%,对未负载TAA的自体靶细胞未见明显杀伤作用(P0.05)。结论:来源于健康志愿者的外周血单个核细胞体外可以成功诱导扩增出TAA-CTL并具有特异性杀伤功能。     

利用超声触发破坏微泡将靶基因传递给左室心肌

背景 :超声触发破坏微泡 (ＵＴＭＤ)方法被认为可作为特定器官的靶基因治疗手段。但是 ,至今仍没有应用此方法使基因成功表达的报导。本研究用于检测如下假说 :内含ＣＭＶ启动子的腺病毒 β半乳糖苷酶基因 (ａｄｃｍｖ -βｇａｌ)的白蛋白微泡经ＵＴＭＤ后 ,ａｄｃｍｖ -βｇａｌ能在鼠心肌细胞中表达。方法 :ａｄｃｍｖ -βｇａｌ粘附于全氟丙烷包裹的微泡上。平均微泡直径为 3 .0± 1.2 μｍ ,浓度为 1.6± 0 .2× 10 9个 /ｍｌ ,每毫升微泡悬浮液中含有 5× 10 9个ａｄｃｍｖ -βｇａｌ。活体试验证实微泡悬浮液中的病毒效价不受超声波频…     

Comparative Ex Vivo Activity of Novel Endoperoxides in Multidrug-Resistant Plasmodium falciparum and P. vivax

The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC(50)s) in both species (median IC(50)s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity.     

超声微泡造影剂介导VEGF基因治疗大鼠心肌缺血的实验性研究

目的：探讨超声微泡造影剂介导血管内皮生长因子（VEGF）基因转染大鼠缺血心肌的有效性。方法：将15只急性心肌梗塞3天后的雄性Wistar大鼠分为3组，每组5只，第一组采用超声破坏微泡造影剂的方式将pcD2VEGF121基因转染大鼠心肌约6分钟；第二组尾静脉输入同等剂量携pcD2VEGF121基因的造影剂；第三组为对照。2周后，分别行免疫组织化学检测缺血心肌中的VEGF蛋白及毛细血管内皮细胞，结果：采用超声微泡造影剂介导的VEGF基因转染，能明显增强VEGF基因在大鼠心肌组织内的表达，并可促进缺血心肌组织新生血管，结论：超声微泡造影剂介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法。     

超声破坏微泡对心肌细胞膜通透性影响的研究

目的探讨超声破坏微泡造影剂对心肌细胞膜通透性的影响。方法将15只昆明小白鼠随机分为3组,一组采用尾静脉输入白蛋白微泡造影剂,胸壁用频率为1MHz,强度为1.5W/cm2的超声进行辐照1min;一组单纯采用同等超声辐照相同时间;一组作为对照。作用后即刻取心肌组织用硝酸镧(La)示踪法做透射电镜观察心肌细胞膜通透性的变化。结果采用超声破坏微泡后可见部分镧颗粒分布于心肌细胞胞浆内,部分分布于线粒体外、细胞核外;单纯超声作用组心肌细胞胞浆中可有少量的镧颗粒分布,而大部分镧颗粒分布于细胞膜外;而对照组镧颗粒分布于心肌细胞膜外。结论超声破坏微泡可提高心肌细胞膜通透性,可能是超声微泡造影剂增强组织中基因转染的机制之一。     

超声破坏微泡促进骨骼肌血管新生的实验研究

目的　探讨超声破坏微泡引起的微血管破裂促进大鼠骨骼肌血管新生的有效性。方法　将 30只 Wistatr大鼠随机分为 3组 ,1组经静脉输入自制的白蛋白微泡造影剂 0 .5 ml,同时用 1MHz,2 .0 W/cm2 的超声在其骨骼肌局部间歇作用 2 min;1组仅在骨骼肌局部用同等的超声能量作用 ;第 3组作为对照组。实验结束后 ,各组分别取 1只做石蜡切片观察显微结构的变化。各组剩余的大鼠于 2周后处死 ,免疫组化观察骨骼肌中血管内皮生长因子 (VEGF)和血管内皮细胞 因子的表达。结果　超声破坏微泡组大鼠骨骼肌中可见 VEGF和 因子的表达较多 ,有较多的新生血管生成 ;而单纯超声作用组 VEGF和 因子表达较少 ,血管新生不明显 ;对照组中无 VEGF和 因子表达 ,无新生血管。结论　超声破坏微泡引起的微血管破裂可刺激骨骼肌中内源性 VEGF的分泌 ,促进血管生成。     

Pharmacokinetics and Ex Vivo Pharmacodynamic Antimalarial Activity of Dihydroartemisinin-Piperaquine in Patients with Uncomplicated Falciparum Malaria in Vietnam

Compared to healthy subjects, malaria patients show a reduction in the mean oral clearance (1.19 versus 5.87 liters/h/kg of body weight) and apparent volume of distribution (1.47 versus 8.02 liters/kg) of dihydroartemisinin in Vietnamese patients following treatment with dihydroartemisinin-piperaquine (Artekin) for uncomplicated Plasmodium falciparum. Dihydroartemisinin is responsible for most of the ex vivo antimalarial activity of dihydroartemisinin-piperaquine.Dihydroartemisinin-piperaquine (Artekin) is an artemisinin-based combination treatment (ACT) drug that is well tolerated and highly effective in the treatment of Plasmodium falciparum malaria in Southeast Asia and Africa, with cure rates typically greater than 95% following a standard 3-day course (8, 9, 15, 17). Despite its extensive use over the past 5 years, no data are available on the clinical pharmacokinetics of dihydroartemisinin in malaria patients following treatment with the ACT. Few studies have investigated the pharmacokinetics of piperaquine in malaria patients. The highly lipophilic drug exhibits biphasic disposition kinetics, with a large apparent volume of distribution, low oral clearance, and a lengthy elimination half-life of about 3 to 4 weeks in malaria patients treated with dihydroartemisinin-piperaquine (6, 18).The present study investigated the clinical pharmacokinetic properties of dihydroartemisinin and piperaquine after a 3-day course of dihydroartemisinin-piperaquine in the treatment of uncomplicated P. falciparum malaria in Vietnamese patients. In addition to assessing the in vivo response of the ACT, the ex vivo pharmacodynamic antimalarial activity of dihydroartemisinin-piperaquine in the patients'' plasma samples was investigated against two lines of P. falciparum. The study was conducted at Military Hospital 175 in Ho Chi Minh City, Vietnam, in 12 adult Vietnamese patients. The volunteers had to satisfy the following inclusion criteria: male, aged 17 to 55 years old, parasite density between 500 and 100,000 per μl of blood, axillary temperature of ≥37.5°C or a history of fever in the previous 24 h, written informed consent, and willing to be monitored for 35 days. The exclusion criteria were as follows: antimalarial treatment within the preceding 2 weeks, mixed plasmodial infection, and history of another serious medical disease. The patients acquired their infections in Binh Phuoc Province, about 120 km from Ho Chi Minh City. The patients stayed at the hospital for the entire 35-day follow-up period and because Ho Chi Minh City is free of malaria, the possibility of reinfection was avoided. Ethical approval for the study was obtained from the Review and Scientific Board of Military Hospital 175 and the Australian Defense Human Research Ethics Committee (ADHREC protocol 379/05).The patients were administered a weight-based 3-day course of dihydroartemisinin-piperaquine (Holleykin Pharmaceuticals, China) (each tablet contained 40 mg of dihydroartemisinin and 320 mg of piperaquine phosphate) at 2.4 mg of dihydroartemisinin per kg of body weight and 19.2 mg of piperaquine per kg of body weight per day, rounded up or down to the nearest half tablet, with day 0 being designated the first dose. Dihydroartemisinin-piperaquine was administered within 15 min of having a standard Vietnamese breakfast of rice, noodles, and meat to enhance the absorption of piperaquine (13). Parasitemia and axillary temperature were measured before commencement of treatment and then every 8 h afterwards to determine parasite and fever clearance times. Blood samples (7 ml) were collected at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 h using an indwelling cannula kept patent with heparinized saline after the last dose of dihydroartemisinin-piperaquine on day 2. Subsequent heparinized blood samples were collected by venipuncture at days 3, 4, 7, 14, 21, 28, and 35 after commencement of treatment. Blood samples were centrifuged, and the separated plasma samples were stored at −25°C until transported on dry ice to Australia for drug analysis, which was within 12 months of collection.Plasma dihydroartemisinin concentrations were measured by liquid chromatography-tandem mass spectrometry, with a lower limit of quantification of 1 ng/ml (2). The values for overall precision of analysis for dihydroartemisinin, as defined by the percent coefficient of variation of spiked samples were 6.3% at 1 ng/ml, 5.7% at 20 ng/ml, 4.6% at 200 ng/ml, and 6.6% at 750 ng/ml. The corresponding inaccuracy values were 1.3%, 1.5%, 0.7%, and 3.2% for 1, 20, 200, and 750 ng/ml, respectively. Plasma piperaquine concentrations were measured by a validated high-performance liquid chromatography method, with a lower limit of quantification of 5 ng/ml (11). The precision values of the assay were 10.3% at 10 ng/ml, 6.8% at 100 ng/ml, 6.7% at 500 ng/ml, and 6.5% at 1,000 ng/ml. The corresponding inaccuracy values were 11.1%, 2.8%, 0.8%, and 0.5% for 10, 100, 500, and 1,000 ng/ml, respectively. Pharmacokinetic parameters (peak concentration [C_max], time to reach maximum concentration [T_max], area under the concentration-time curve from day 2 to the last data point [AUC_d2-ld] and from day 2 to infinity [AUC_d2-∞], terminal half-life [t_1/2], apparent oral clearance, and apparent volume of distribution) were determined from the plasma concentration-time data using noncompartmental methods.The ex vivo pharmacodynamic antimalarial activity of dihydroartemisinin-piperaquine was assessed by culturing malaria parasites in vitro in the presence of patients'' plasma samples collected after the last administration of dihydroartemisinin-piperaquine by the method of Kotecka et al. (10), with minor modifications. Briefly, patients'' plasma samples (50 μl) were serially diluted twofold on microtiter plates with drug-free human plasma. The in vitro drug susceptibility of two lines of P. falciparum (chloroquine-sensitive D6 and chloroquine-resistant K1) to dihydroartemisinin and piperaquine were determined in parallel with the patients'' plasma samples. Fifty microliters of spiked drug solutions prepared in human plasma were serially diluted twofold on microtiter plates using drug-free plasma. Inoculum (50 μl) was added to each well containing either the patient''s plasma samples or spiked drug solutions, so that the total cell suspension (100 μl) contained 50% plasma in hypoxanthine-free plain LPLF-RPMI 1640, with a final hematocrit of 2% and a parasitemia (>95% rings) of 1%. Tritiated hypoxanthine incorporation was used to determine the extents to which parasite growth was inhibited by different drug concentrations or dilutions of the patients'' plasma during 48 h of incubation. The inhibitory concentration (90% infective concentration [IC₉₀] for spiked drug samples) and inhibitory dilution (90% infective dose [ID₉₀] for patient plasma samples) were defined as the drug concentrations and the number of dilutions of the patient plasma sample, respectively, that produced a 90% inhibition of uptake of tritiated hypoxanthine by intraerythrocytic malaria parasites compared to drug-free plasma samples (controls).The mean (standard deviation) age of the patients was 26.3 (10.4) years, with a mean (standard deviation) weight of 56.5 (7.8) kg. The patients had an admission geometric mean parasitemia of 15,198 parasites/μl of blood (range, 738 to 79,310 parasites/μl) and a mean temperature (standard deviation) of 37.7°C (1.3°C), with 42% (5 of 12) of patients with fever. Treatment with dihydroartemisinin-piperaquine promptly reduced fever, with a median fever clearance time of 24 h (range, 16 to 48 h) and led to a rapid reduction in parasite density, with a median parasite clearance time of 28 h (range, 16 to 56 h). Over the 35-day follow-up period, there was no recurrence of infection in the patients. The mean content values of five dihydroartemisinin-piperaquine tablets were 105.4% ± 4.8% for dihydroartemisinin and 109.0% ± 1.1% for piperaquine. Although none of the patients reported treatment with antimalarial drugs 2 weeks before commencing dihydroartemisinin-piperaquine, no dihydroartemisinin or piperaquine was detected in their predose samples, which confirmed no recent treatment with either artesunate or CV8 (dihydroartemisinin-piperaquine-primaquine-trimethoprim).The mean plasma concentration-time profiles of dihydroartemisinin and piperaquine after a 3-day course of dihydroartemisinin-piperaquine are shown in Fig. ​Fig.1,1, and the pharmacokinetics of the two drugs are summarized in Table ​Table1.1. Because dihydroartemisinin is rapidly eliminated with a t_1/2 of about 1 h in healthy Vietnamese subjects (2) and does not accumulate with daily administration, we were able to compare the pharmacokinetics of dihydroartemisinin in the Vietnamese patients after the last daily dose of the 3-day course of dihydroartemisinin-piperaquine with values obtained in healthy Vietnamese subjects given a single dose of dihydroartemisinin-piperaquine (2). The mean C_max and AUC of dihydroartemisinin were markedly higher in the Vietnamese malaria patients than in the healthy Vietnamese subjects (C_max, 698 versus 176 ng/ml; AUC_d2-∞, 1,949 versus 398 ng·h/ml). Although the difference was not as large, Binh et al. (1) reported an approximately twofold-higher mean C_max (1,045 versus 480 ng/ml) and AUC (2,401 versus 932 ng·h/ml) of dihydroartemisinin in Vietnamese malaria patients compared with healthy subjects administered a single dose of dihydroartemisinin (120 mg) alone. Dihydroartemisinin was rapidly eliminated in the malaria patients with a t_1/2 of 0.85 ± 0.15 h. The apparent oral clearance and apparent volume of distribution of dihydroartemisinin were 4.9-fold lower (1.19 versus 5.87 liters/h/kg) and 5.5-fold lower (1.47 versus 8.02 liters/kg) in the Vietnamese malaria patients than in the healthy subjects, respectively (2). A reduction in clearance and contraction in the apparent volume of distribution has also been reported for other antimalarial drugs, such as mefloquine (7) and quinine (21), during the acute phase of malaria. A likely explanation for the increase in bioavailability of dihydroartemisinin in malaria patients compared with healthy subjects is a decrease in hepatic clearance of dihydroartemisinin due to malaria (1). Alpha-acid glycoprotein levels also increase during acute malaria (16), and similar to quinine, this may cause an increase in binding of the protein-bound dihydroartemisinin, with a reduction in the apparent volume of distribution of the drug (12).Open in a separate windowFIG. 1.Plasma dihydroartemisinin (○) and piperaquine (•) concentration-time profiles after the last dose of a 3-day course of dihydroartemisinin-piperaquine (2.4 mg of dihydroartemisinin per kg and 19.2 mg of piperaquine per kg daily) for the treatment of uncomplicated Plasmodium falciparum malaria in 12 Vietnamese patients. The values shown are means plus standard deviations (error bars). The inset shows the piperaquine concentrations from day 2 to day 35 after commencement of treatment. The ex vivo pharmacodynamic antimalarial activity profile (mean ID₉₀ values ▴) of patients'' plasma samples after dihydroartemisinin-piperaquine treatment is derived from the K1 line of P. falciparum.

TABLE 1.

Pharmacokinetic parameters of dihydroartemisinin and piperaquine in 12 Vietnamese patients with uncomplicated P. falciparum malaria^a

超声微泡介导EGFP转染正常大鼠关节滑膜组织的研究

目的 探讨超声微泡介导EGFP转染大鼠关节滑膜组织的可能性.方法 24只正常清洁级Wister大鼠;4只为单纯对照组,20只为实验组.实验组根据质粒的剂量又分4组,每组5只(10个膝关节),1组:超声+微泡+1μg EGFP;2组:超声+微泡+5μgEGFP;3组:超声+微泡+10μg EGFP;4组:超声+微泡+15μg EGFP.将300μl SonoVue与EGFP混合,注射入大鼠关节腔内,超声持续照射10 min.3 d后取大鼠膝关节滑膜组织,直接贴在载玻片上,在荧光显微镜480 nm波长激发状态下观察EGFP转染效果.结果 1μg和5μg EGFP组与单纯对照组比较,荧光强度稍有增强,但是不明显;而10μg和15μg组荧光强度与单纯对照组比较均有明显增强.结论 超声介导微泡可以实现EGFP质粒对正常大鼠关节滑膜组织的转染,10μg EGFP是本实验的最佳剂量.     

电镜硝酸镧示踪超声微泡造影剂开放血脑屏障

目的 研究超声波破坏微泡造影剂对大鼠血脑屏障通透性的影响。 方法 用电镜硝酸镧示踪法观察超声波破坏微泡造影剂对大鼠血脑屏障通透性的变化。 结果 超声波照射后即刻，即可见镧颗粒通过毛细血管内皮细胞及细胞间的紧密连接进入组织间隙，血脑屏障开放持续至6h，12h时已关闭。电镜下可以见到血管源性脑水肿，细胞器水肿不明显。 结论 超声波破坏微泡造影剂开放血脑屏障，为中枢神经系统疾病的治疗提供了一种无创、具靶向性的药物转运方法。