Pre-clinical pharmacokinetics of novel soybean iosflavone sulfonate

为寻找新的大豆异黄酮前药,采用建立的生物样品中药物浓度测定的液相色谱法对新型大豆异黄酮染料木素磺酸酯(GBS)进行前药判定以及大鼠体内药物动力学研究,以考察前药中染料木素(GE)的口服相对生物利用度是否改善.在大鼠体内药物代谢实验中,灌胃给予的大鼠血浆中能检测到GE的存在.在临床前药物动力学实验中,该前体药以40 mg/kg GE在大鼠体内的动力学过程符合一室模型.GBS中GE的相对口服生物利用度为原药的198.6％.结果表明:相对于原药GE,前药中GE的相对口服生物利用度得到极大地改善.该前药有进一步研究的意义.     

丁香花蕾油作为微乳的油相促进黄芩苷透皮吸收的研究

目的:黄芩苷作为黄芩的主要成分之一,其制剂虽应用广泛,但存在较多缺点,如水溶液不稳定、口服吸收差、生物利用度不高及药物体内半衰期短等从而导致用药剂量大且频繁给药。本文目的是研究丁香花蕾精油及其微乳对黄芩苷体外经皮渗透性的影响。方法:采用Valia-Chien扩散池,以生理盐水为接受液,不同浓度的丁香花蕾精油以及以丁香花蕾精油作为微乳的油相及促进剂进行大鼠离体皮肤的体外渗透实验。结果:黄芩苷微乳的平均粒径为41.3±5.1 nm,多分散指数为0.18,表明该制剂粒径小且均一。微乳(不含丁香花蕾精油)、丁香花蕾精油及含丁香花蕾精油的微乳对黄芩苷均有不同程度的增渗作用。其中,含丁香花蕾精油的微乳对黄芩苷的促渗作用最强。结论:丁香花蕾精油以及以丁香花蕾精油作为微乳的油相对黄芩苷均有一定的促进渗透作用。     

新型大豆异黄酮磺酸酯的临床前药物动力学

为寻找新的大豆异黄酮前药,采用建立的生物样品中药物浓度测定的液相色谱法对新型大豆异黄酮染料木素磺酸酯(GBS)进行前药判定以及大鼠体内药物动力学研究,以考察前药中染料木素(GE)的口服相对生物利用度是否改善。在大鼠体内药物代谢实验中,灌胃给予的大鼠血浆中能检测到GE的存在。在临床前药物动力学实验中,该前体药以40mg/kg GE在大鼠体内的动力学过程符合一室模型。GBS中GE的相对口服生物利用度为原药的198.6%。结果表明:相对于原药GE,前药中GE的相对口服生物利用度得到极大地改善。该前药有进一步研究的意义。     

新型染料木素磺酸酯的前药判定与大鼠体内生物利用度

为寻找染料木素(GE)新的前药,采用建立的生物样品中药物浓度测定的液相色谱法对新型大豆异黄酮染料木素磺酸酯(GB)进行前药判定以及大鼠体内药物动力学研究,以考察前药中染料木素的口服生物利用度是否改善.在大鼠体内药物代谢实验中,灌胃给予GB的大鼠血浆中能检测到明显的GE.在临床前药物动力学实验中,该前体静注给药和以40 mg/kg灌胃药后,GE在大鼠体内的动力学过程均符合一室模型.GB中GE的相对口服生物利用度为原药的110.9％.研究表明,相对于原药GE,前药中GE的相对口服生物利用度达到预期的改善,该前药有进一步研究意义.     

β-榄香烯对卵巢癌细胞SKOV3增殖和凋亡的影响

目的:探讨β-榄香烯对卵巢癌细胞SKOV3增殖和凋亡的影响。方法:体外培养卵巢癌SKOV3细胞,将β-榄香烯(浓度梯度为25,50,100,150,200μg/m L),单独作用于卵巢癌SKOV3细胞,加药24 h、48 h后用噻唑蓝(MTT法)法检测细胞增殖情况;用流式细胞术检测加药24 h后对细胞凋亡的影响。结果:1MTT法结果显示β-榄香烯单独用药24 h、48 h后,与对照组相比,实验组对卵巢癌SKOV3细胞的抑制率均高于对照组(P0.05),并且在一定程度上呈浓度和时间依赖性。2流式细胞术检测显示,β-榄香烯能够促进SKOV3细胞的凋亡。结论:β-榄香烯能够抑制卵巢癌细胞SKOV3的增殖,促进其凋亡。     

beta-榄香烯对卵巢癌细胞SKOV3 增殖和凋亡的影响

目的:探讨β-榄香烯对卵巢癌细胞SKOV3增殖和凋亡的影响。方法:体外培养卵巢癌SKOV3细胞,将β-榄香烯(浓度梯度为25,50,100,150,200μg/m L),单独作用于卵巢癌SKOV3细胞,加药24 h、48 h后用噻唑蓝(MTT法)法检测细胞增殖情况;用流式细胞术检测加药24 h后对细胞凋亡的影响。结果:1MTT法结果显示β-榄香烯单独用药24 h、48 h后,与对照组相比,实验组对卵巢癌SKOV3细胞的抑制率均高于对照组(P<0.05),并且在一定程度上呈浓度和时间依赖性。2流式细胞术检测显示,β-榄香烯能够促进SKOV3细胞的凋亡。结论:β-榄香烯能够抑制卵巢癌细胞SKOV3的增殖,促进其凋亡。     

抗癌药物榄香烯的生物合成及关键酶吉马烯A合酶的优化

榄香烯是我国拥有自主知识产权的抗肿瘤药物之一,因其抗肿瘤活性强、作用范围广、毒副作用轻微和不易产生耐药性等优点,被广泛应用于各类恶性肿瘤的临床治疗中。榄香烯的生产主要依靠药用植物温郁金的分离提取。但温郁金的榄香烯含量低、分离纯化难度大、得率低且成本高,严重阻碍了榄香烯的大规模生产与应用。随着合成生物学的发展,利用微生物构建细胞工厂用于生物合成天然药物成为研究热点,也为榄香烯的生产提供了新的思路。近年来,对榄香烯的生物合成研究在不断深入。研究者通过代谢工程、组合生物学和基因工程等手段,阐明榄香烯生物合成途径和关键酶,已经成功克隆了榄香烯生物合成途径上的一些关键酶基因,初步实现榄香烯的异源生物合成。本文以合成生物学研究思维概述榄香烯生物合成途径及其工程菌的优化,重点综述关键酶吉马烯A合酶(germacrane A synthase, GAS)。从限速酶基因的过表达和分流基因的敲除,融合表达酶工程策略、吉马烯A合酶的体外进化几方面,对其生物合成途径的改造策略进行阐述。同时,也分析提高异源生物合成榄香烯产量所面临的问题与挑战,为榄香烯的高效生物合成提供参考。     

青藤碱磷脂复合物纳米结构脂质载体的制备、表征及药动学研究

为制备青藤碱磷脂复合物纳米结构脂质载体,并进行体外和SD大鼠体内评价。实验采用溶剂挥发法制备青藤碱磷脂复合物,乳化超声法制备青藤碱磷脂复合物纳米结构脂质载体。考察其粒径分布、Zeta电位,包封率,载药量及体外释药等基本理化性质。SD大鼠分别灌胃给予青藤碱混悬液和青藤碱磷脂复合物纳米结构脂质载体,比较药动学行为及生物利用度。结果显示,青藤碱磷脂复合物纳米结构脂质载体的平均粒径为201.32±5.05 nm,Zeta电位为-22.2±1.5 mV,包封率为80.31±1.01%,载药量为4.42±0.28%,体外释药具有明显的缓释特征,体外释药模型符合Weibull释药模型,拟合方程为:LnLn(1/1-Mt/M∞)=0.576 6Lnt-1.478 1(r=0.988 8)。体内药动学研究结果表明,磷脂复合物纳米结构脂质载体改变了青藤碱的药动学行为,增强了体内吸收,延长了青藤碱在体内滞留时间,相对生物利用度提高到了1.75倍。因此,青藤碱磷脂复合物纳米结构脂质载体可显著促进青藤碱体内吸收,提高其口服生物利用度。     

非诺贝特固体自乳化制剂（S-SEDDS）体外评价及生物利用度研究

目的:以S-SEDDS替代液态自乳化制剂中的表面活性剂制备非诺贝特固体自乳化制剂。方法:比较了固体自乳化制剂与市售产品、液体自乳化制剂的体外溶出情况及体内生物利用度。结果:表明本研究的固体自乳化制剂用水分散后平均粒径为820.2±26.5(nm);溶出度试验结果显示,30min累积溶出度达到80%以上,本研究制备的非诺贝特固体自乳化制剂AUC(0-24)为22.7±8.2 mgoL-1oh与SEDDS的AUC(0-24)(24.9±7.6mgoL-1oh)没有显著性差异(P>0.05),与市售微粉化胶囊(13.8±10.5mgoL-1oh)相比能够显著提高药物的生物利用度(P<0.05)。结论:S-SEDDS有液体自乳化给药系统的效果。     

高乌甲素磷脂复合物纳米粒的制备、表征及药动学研究

采用溶剂挥发法制备高乌甲素磷脂复合物,薄膜超声法制备高乌甲素磷脂复合物固体脂质纳米粒,并考察基本理化性质。SD大鼠分别灌胃给予高乌甲素及其磷脂复合物固体脂质纳米粒,比较药动学特征及生物利用度。结果表明,高乌甲素磷脂复合物固体脂质纳米粒平均粒径为(224.37±1.93)nm,Zeta电位为(-6.14±0.09)m V,包封率为(85.08±1.84)%。体外释药符合Weibull模型。药动学结果显示,Tmax,Cmax,AUC0~t和AUC0~?均具有统计学意义(P0.05或P0.01),口服吸收生物利用度提高了2.58倍。因此,高乌甲素磷脂复合物固体脂质纳米粒可有效提高高乌甲素的口服吸收生物利用度。     

Design and development of microemulsion drug delivery system of acyclovir for improvement of oral bioavailability

The main purpose of this work was to develop an oral microemulsion formulation for enhancing the bioavailability of acyclovir. A Labrafac-based microemulsion formulation with Labrasol as surfactant and Plurol Oleique as cosurfactant was developed for oral delivery of acyclovir. Phase behavior and solubilization capacity of the microemulsion system were characterized, and in vivo oral absorption of acyclovir from the microemulsion was investigated in rats. A single isotropic region, which was considered to be a bicontinuous microemulsion, was found in the pseudoternary phase diagrams developed at various Labrasol:Plurol Oleique:Labrafac ratios. With the increase of Labrasol concentration, the microemulsion region area and the amount of water and Labrafac solubilized into the microemulsion system increased; however, the increase of Plurol Oleique percentage produced opposite effects. The microemulsion system was also investigated in terms of other characteristics, such as interfacial tension, viscosity, pH, refractive index, diffusion, and bioavailability. Acyclovir, a poorly soluble drug, displayed high solubility in a microemulsion formulation using Labrafac (10%). Labrasol (32%), Plurol Oleique (8%), and water (50%). The in vitro intraduodenal diffusion and in vivo study revealed an increase of bioavailability (12.78 times) after oral administration of the microemulsion formulation as compared with the commercially available tablets. Published: September 15, 2006     

Evidence that renal arginine transport is impaired in spontaneously hypertensive rats

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired l-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the l-arginine transport inhibitor l-lysine (10 μmol·kg(-1)·min(-1); 30 min) and subsequent superimposition of l-arginine (100 μmol·kg(-1)·min(-1); 30 min), the NO synthase inhibitor N(G)-nitro-l-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 μg·kg(-1)·min(-1)) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). l-Lysine tended to reduce medullary perfusion (-15 ± 7%; P = 0.07) and reduced medullary NO concentration (-9 ± 3%; P = 0.03) while subsequent superimposition of l-arginine reversed these effects of l-lysine in SD rats. In SHR, l-lysine and subsequent superimposition of l-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal l-arginine transport is impaired in SHR. Renal l-[(3)H]arginine transport was less in SHR compared with SD rats (P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney.     

Enhanced Intestinal Absorption of Daidzein by Borneol/Menthol Eutectic Mixture and Microemulsion

In the present study, the effect of a borneol/menthol eutectic mixture (25:75) and microemulsion on the absorption of daidzein in rat intestinal membrane was evaluated. The microemulsion formulation was composed of ethyl oleate (oil), Cremophor RH40 (surfactant), PEG400 (co-surfactant), and water. The borneol/menthol eutectic mixture and its microemulsion were found to enhance the intestinal absorption of daidzein in vitro. A diffusion chamber system with isolated rat intestinal membranes was used. In contrast, verapamil (0.3 mM), a typical P-glycoprotein inhibitor, showed no effect on the absorption of daidzein by this system. A pharmacokinetic study was conducted in rats. After oral administration of daidzein at a dose of 10 mg/kg in the form of either borneol/menthol eutectic mixtures or suspension, the relative bioavailability of borneol/menthol eutectic mixtures and microemulsion was enhanced by about 1.5- and 3.65-fold, respectively, compared with a daidzein suspension. In conclusion, a borneol/menthol eutectic mixture can enhance the absorption of daidzein, although the mechanism of absorption enhancement is still unclear.     

Role of cytochrome P-450 metabolites in the regulation of renal function and blood pressure in 2-kidney 1-clip hypertensive rats

Alterations in renal function contribute to Goldblatt two-kidney, one-clip (2K1C) hypertension. A previous study indicated that bioavailability of cytochrome P-450 metabolites epoxyeicosatrienoic acids (EETs) is decreased while that of 20-hydroxyeicosatetraenoic acids (20-HETE) is increased in this model. We utilized the inhibitor of soluble epoxide hydrolase cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB) and HET-0016, the inhibitor of 20-HETE production, to study the role of EETs and 20-HETE in the regulation of renal function. Chronic c-AUCB treatment significantly decreased systolic blood pressure (SBP) (133 ± 1 vs. 163 ± 3 mmHg) and increased sodium excretion (1.23 ± 0.10 vs. 0.59 ± 0.03 mmol/day) in 2K1C rats. HET-0016 did not affect SBP and sodium excretion. In acute experiments, renal blood flow (RBF) was decreased in 2K1C rats (5.0 ± 0.2 vs. 6.9 ± 0.2 ml·min(-1)·g(-1)). c-AUCB normalized RBF in 2K1C rats (6.5 ± 0.6 ml·min(-1)·g(-1)). HET-0016 also increased RBF in 2K1C rats (5.8 ± 0.2 ml·min(-1)·g(-1)). Although RBF and glomerular filtration rate (GFR) remained stable in normotensive rats during renal arterial pressure (RAP) reductions, both were significantly reduced at 100 mmHg RAP in 2K1C rats. c-AUCB did not improve autoregulation but increased RBF at all RAPs and shifted the pressure-natriuresis curve to the left. HET-0016-treated 2K1C rats exhibited impaired autoregulation of RBF and GFR. Our data indicate that c-AUCB displays antihypertensive properties in 2K1C hypertension that are mediated by an improvement of RBF and pressure natriuresis. While HET-0016 enhanced RBF, its anti-natriuretic effect likely prevented it from producing a blood pressure-lowering effect in the 2K1C model.     

头孢氨苄胶囊制剂人体生物等效性研究

目的:研究新仿制的头孢羟氨苄胶囊制剂与市场在售的同类制剂在健康人体内的生物等效性。方法:采用随机交叉试验设计,20名健康男性志愿者分别口服受试制剂与参比制剂500 mg,HPLC法测定血浆中头孢氨苄的浓度,用DAS 2.0软件计算药动学参数并进行生物等效性评价。结果:受试制剂和参比制剂两药的主要药代动力学参数,Cmax分别为(18.12±3.17)μg/ml和(21.28±3.77)μg/ml,Tmax分别为(1.09±0.37)h和(1.04±0.33)h,t1/2分别为(1.15±0.22)h和(1.14±0.20)h,AUC0-t分别为(35.43±5.39)μg h/ml和(37.27±4.76)μg h/ml,AUC0-∞分别为(36.68±6.06)μg h/ml和(38.62±5.48)μg h/ml,受试制剂的平均相对生物利用度为(96.50±7.2)%。结论:受试制剂和参比制剂具有良好的生物等效性。     

三氧化二砷对人脐静脉内皮细胞凋亡及VCAM-1/ICAM-1 表达的影响

目的:观察三氧化二砷(As2O3)对血管内皮细胞增殖、凋亡及VCAM-1/ICAM-1表达的影响,探讨As2O3对血管内皮细胞增殖生长以及炎症反应的影响。方法:人脐静脉内皮细胞(HUVEC)体外培养,以不同As2O3浓度及时间对其进行干预。采用CCK-8测定细胞增殖活性,流式细胞仪AnnexinⅤ/PI双染法检测细胞的凋亡率,实时荧光定量PCR检测VCAM-1mRNA表达,酶联免疫吸附试验(ELISA)检测细胞间黏附分子(VCAM-1)及血管细胞黏附分子(ICAM-1)的表达情况。结果:当As2O3浓度在3μmol.L-1时HUVEC培养24 h的的凋亡率为(0.134±0.03)%,48 h为(3.305±0.53)%,72 h为(3.748±0.84)%(P<0.05),凋亡率均在一较低水平。当As2O3浓度>3μmol.L-1时HUVEC凋亡率明显增加(P<0.01)。不同浓度As2O3作用HUVEC48 h后检测上清液中ICAM-1与VCAM-1浓度时发现1μmol.L-1时VCAM-1表达即开始增加(123.32±3.78 mmol.L-1,P<0.01),而HUVEC表达ICAM-1含量与对照组相比差异并不明显(38.94±2.59 mmol.L-1,P>0.05),随着As2O3浓度的增加,HUVEC表达ICAM-1/VCAM-1的量均增加但敏感性不同。对照组及(1.0、2.0、3.0、4.0、5.0)μmol.L-1As2O3作用于HUVEC 48 h实时荧光定量PCR法检测VCAM-1mRNA表达量明显增加,与对照组相比实验组的表达量分别为(1.657±0.287,1.858±0.241,2.321±0.280,3.012±0.235,3.508±0.342)(P<0.01)。结论:As2O3可直接降低细胞活性,诱导细胞凋亡,并且呈一定的时间-浓度依赖性。在较低浓度时VCAM-1/ICAM-1的表达在一个相对较低的水平,随着As2O3浓度的逐渐升高,内皮细胞凋亡率增高,VCAM-1/ICAM-1表达增加,并且VCAM-1/ICAM-1对As2O3的敏感性呈现一定的差异性。     

Intranasal Microemulsion of Sildenafil Citrate: In Vitro Evaluation and In Vivo Pharmacokinetic Study in Rabbits

The purpose of the present study was to prepare intranasal delivery system of sildenafil citrate and estimate its relative bioavailability after nasal administration in rabbits to attain rapid onset of action with good efficacy at lower doses. Sildenafil citrate saturated solubility was determined in different solvents, cosolvents, and microemulsion systems. For nasal application, sildenafil citrate was formulated in two different systems: the first was a cosolvent system (S3) of benzyl alcohol/ethanol/water/Transcutol/taurodeoxy cholate/Tween 20 (0.5:16.8:47.7:15.9:1:18.1% w/w). The second was a microemulsion system (ME6) containing Oleic acid: Labrasol/Transcutol/water (8.33:33.3:16.66:41.66% w/w). The prepared systems were characterized in relation to their clarity, particle size, viscosity, pH, and nasal ciliotoxicity. In vivo pharmacokinetic performance of the selected system ME6 (with no nasal ciliotoxicity) was evaluated in a group of six rabbits in a randomized crossover study and compared to the marketed oral tablets. The targeted solubility (>20 mg/ml) of sildenafil citrate was achieved with cosolvent systems S1, S3, and S5 and with microemulsion systems ME3–ME6. The saturated solubility of sildenafil citrate in cosolvent system S3 and microemulsion system ME6 were 22.98 ± 1.26 and 23.79 ± 1.16 mg/ml, respectively. Microemulsion formulation ME6 showed shorter t _max (0.75 h) and higher AUC_(0-∞) (1,412.42 ng h/ml) compared to the oral tablets which showed t _max equals 1.25 h and AUC_(0-∞) of 1,251.14 ng h/ml after administration to rabbits at dose level of 5 mg/kg. The relative bioavailability was 112.89%. In conclusion, the nasal absorption of sildenafil citrate microemulsion was found to be fast, indicating the potential of nasal delivery instead of the conventional oral administration of such drug.     

孟鲁斯特钠咀嚼片在健康人体内药动学和生物等效性

目的:研究两种(仿制新药与市售)孟鲁司特钠咀嚼片在人体内生物等效性。方法:采用单中心、随机、开放、双周期自身交叉试验设计,20名健康男性志愿者分2周期分别口服受试制剂和参比制剂各10 mg,HPLC法测定血浆中孟鲁司特钠咀嚼片的浓度,用DAS2.1.1软件计算人体药动学参数并进行生物等效性评价。结果:受试制剂和参比制剂两药的主要药代动力学参数AUC0-t分别为(17.94±6.19)μg h/ml和(17.37±4.73)μg h/ml,AUC0-∞分别为(18.26±6.16)μg h/ml和(17.64±4.66)μg h/ml,Cmax分别为(5.58±1.95)μg/ml和(5.54±1.65)μg/ml,Tmax分别为(2.03±0.97)h和(1.93±0.69)h,t1/2分别为(1.20±0.17)h和(1.19±0.13)h。受试制剂的平均相对生物利用度为(101.5±6.56)%。结论:受试制剂和参比制剂具有生物等效性。     

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells

Abstract

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion - exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.     

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.