Disposition Characteristics of Plasmid DNA in the Single-pass Rat Liver Perfusion System

Purpose. To define the hepatic uptake mechanism of a plasmid DNA, we quantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene fused to simian virus 40 promoter), a model plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4). Methods. [³²P]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the outflow patterns and hepatic uptake were evaluated using statistical moment analysis. Results. The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasmid is fairly stable in the perfusate during liver perfusion. In bolus experiments, pCAT was largely taken up by the liver and the uptake was decreased with increase in injected dose. Statistical moment analysis against outflow patterns demonstrated that the apparent volume of distribution of pCAT was greater than that of human serum albumin, indicating a significant reversible interaction with the tissues. The results of collagenase perfusion experiments suggest that the hepatic accumulation of pCAT occurred preferentially in the nonparenchymal cells (NPC). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, succinylated bovine serum albumin, but not by polycytidylic acid. This suggests that pC AT is taken up by the liver via scavenger receptors for polyanions on the NPC. In constant infusion experiments, the presence of 2,4-dinitrophenol and NH₄C1 caused a significant increase in the outflow concentration of [³²P]pCAT and decrease by half in the total hepatic recovery than that of plasmid DNA administered alone, suggesting that plasmid DNA may undergo internalization by the NPC. Conclusions. The liver plays an important role in the elimination of plasmid DNA and a successful delivery system will be required to avoid its recognition by the scavenger receptors on the liver NPC.     

Analysis of Hepatic Disposition of Galactosylated Cationic Liposome/Plasmid DNA Complexes in Perfused Rat Liver

Purpose. To determine the intrahepatic disposition characteristics of galactosylated liposome/plasmid DNA (pDNA) complexes in perfused rat liver. Methods. Galactosylated liposomes containing N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cholesterol (Chol), and cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiogalactosylethyl)amino]butyl} formamide (Gal-C4-Chol) were prepared. The liposome/[³²P]-labeled pDNA complexes were administered to perfused liver, and the venous outflow patterns were analyzed based on a two-compartment dispersion model. Results. The single-pass hepatic extraction of pDNA complexed with DOTMA/Chol/Gal-C4-Chol liposomes was greater than that with control DOTMA/Chol liposomes. A two-compartment dispersion model revealed that both the tissue binding and cellular internalization rate were higher for the DOTMA/Chol/Gal-C4-Chol liposome complexes compared with the control liposome complexes. The tissue binding was significantly reduced by the presence of 20 mM galactose. When their cellular localization in the perfused liver at 30 min postinjection was investigated, it was found that the parenchymal uptake of the DOTMA/Chol/Gal-C4-Chol liposome complexes was greater than that of the control liposome complexes. The parenchymal cell/nonparenchymal cell uptake ratio was as high as unity. Conclusion. Galactosylation of the liposome/pDNA complexes increases the tissue binding and internalization rate via an asialoglycoprotein receptor-mediated process. Because of the large particle size of the complexes (150 nm), however, penetration across the fenestrated sinusoidal endothelium appears to be limited.     

三尖杉酯碱脂质体在兔体内的药物动力学

本文比较了游离三尖杉酯碱(free harringtonine,FH)和其高、低包裹率脂质体(high and low en-capsulation percentage harringtonine liposmes,En％HL)在兔体内的药物动力学行为,ⅳ FH,low En％HL和high En％HL的药时曲线均符合双室开放模型,FH,low En％HL和high En％HL的α,β两相的生物半衰期分别为:1.32±0.24,32±6 min;4.12±0.25,106±5 min;9.4±1.2,209±5 min。     

A Scalable,Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

No HeadingPurpose. A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.Methods. Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.Results. Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.Conclusions. This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.     

超氧化物歧化酶脂质体在大鼠体内的药代动力学和组织分布

目的考察3种超氧化物歧化酶(SOD)脂质体静脉给药后在大鼠体内的药代动力学和组织分布。方法 用反相蒸发法制备SOD脂质体，采用黄嘌呤氧化酶法检测SOD活力，静脉注射给药后，测定大鼠血中SOD含量变化和不同组织中SOD含量变化。结果在血浆中，SOD水溶液、SOD普通脂质体、用DSPE-PEG2000修饰的SOD脂质体、用Tween 80修饰的SOD脂质体的半衰期分别为0.25，0.34，0.66和0.41 h；AUC分别为12.48，24.66，41.16和33.02 μg·h·mL^-1。与普通脂质体比较，经过DSPE-PEG和Tween 80修饰后的脂质体，使肝、脾中SOD的含量有不同程度的降低，脑中含量有所提高。结论3种SOD脂质体均可不同程度地延长SOD的血浆半衰期，并以用DSPE-PEG2000修饰的SOD脂质体效果最好。与普通脂质体相比，用Tween 80修饰的SOD脂质体可以提高进入脑中的SOD量，用DSPE-PEG2000修饰的SOD脂质体可以减少肝脾对SOD的摄取。     

利巴韦林脂质体在大鼠体内的药物动力学及其生物利用度

应用高效液相色谱法 ,以市售利巴韦林口服液为对照 ,测定了利巴韦林脂质体灌胃给药后大鼠血清中的药物浓度 ,按非隔室模型计算利巴韦林脂质体的药物动力学参数 ,AUC值为1 1 1 2 6μg·h/mL,MRT值为 2 1 6h ,K值为 0 0 61 /h ,其相对生物利用度为 1 2 1 3 %     

Pharmacokinetics of Intranasally-Administered Dihydroergotamine in the Rat

Intranasal dosing of dihydroergotamine (DHE) allows convenient self-administration and provides an alternate route of administration for the treatment of migraine in addition to the existing parenteral dosage forms. In this study, the pharmacokinetics of ³H-DHE were investigated following intravenous and intranasal dosing (0.343 mg DHE/animal) in the rat. Intranasal administration of DHE resulted in rapid absorption. The extent of absorption of the radiolabeled dose was approximately 45%–60%. Absolute bioavailability of the parent drug was 35%–40%, as determined by deconvolution and by the ratios of AUC_0– following intranasal and intravenous dosing. Due to the limited capacity of the nostrils, approximately half of the intranasal dose was swallowed into the gastrointestinal tract. Biliary excretion was found to be the predominant pathway of radioactivity excretion following both routes of administration. The results from this study suggest that intranasal administration provides a viable means of delivering DHE into the systemic circulation.     

Pharmacokinetics of lopromide Liposomes in Rabbits

Purpose. The dose-proportionality of pharmacokinetics of an iodinated contrast medium, iopromide, encapsulated into liposomes was investigated. Methods. Following single intravenous administration of 150 mg iodine/kg (potential diagnostic dose) and a five-fold higher dose in rabbits the pattern of elimination was studied until 7 d and the blood concentrations were monitored up to 72 h after administration. The iodine concentration in the liver was calculated on the basis of the blood concentration and related to the concentration measured in the rabbit liver. Results. The dose-normalized blood concentration-time profiles of the encapsulated iodine were not superimposable. Contrary to the low dose a steady-state concentration of 2.8 mg iodine/mL was observed in blood for 60 min after the high dose administration indicating a saturation of the liposomal liver uptake. For both doses the elimination of iodine occurred predominantly via the kidneys and was complete 7 d after administration. The dose-normalized amounts of iodine excreted with the urine were similar for both dose groups. From the blood data it was calculated that doses up to about 300 mg iodine/kg should result in a dose-proportional increase of liposomal liver uptake before saturation occurs. This was confirmed by the measured iodine liver concentrations after increasing the doses stepwise from 150 to 750 mg iodine/kg. Conclusions. In rabbits for the dose range 150 to 750 mg iodine/kg iopromide liposomes reveal dose-dependent pharmacokinetics due to a saturation in liver uptake which occurs for doses of 300 mg iodine/kg corresponding to 300 mg lipid/kg onwards.     

替加氟磁性长循环脂质体在大鼠体内的药代动力学和靶向性

目的研究替加氟磁性长循环脂质体经肝动脉给药的药代动力学和组织靶向性。方法采用高效液相色谱仪检测大鼠生物样品中替加氟的浓度。结果磁控并加热的替加氟磁性长循环脂质体组的肝内8h药时曲线下面积(AUC)是游离替加氟组的17.4倍,是替加氟长循环脂质体组的3.9倍;其肝外组织血浆和肾的AUC比游离替加氟组低。其肝靶向效率达到73.9%。结论替加氟磁性长循环脂质体经肝动脉给药能显著增加药物的肝脏靶向性,可能降低其肾毒性。     

Pharmacokinetics and tissue distribution of liposomal etoposide in rats

Precipitation of etoposide and adverse events associated with the co-solvents in intravenous solutions can be avoided by using liposomal etoposide (LE). The pharmacokinetics and distribution of the commercial formulation (ETPI) and LE were compared in rats. The pharmacokinetic profiles were biphasic and similar in the initial phase (C_max, Vd, and t_1/2α). However, LE showed a 60% increase in AUC with a 35% decrease in clearance (p?<?0.05). This decreased clearance resulted in a 70% increase in the MRT of etoposide. The uptake of etoposide from LE was higher in macrophage-phagocytic endowed tissues indicating that LE is superior to ETPI for targeted delivery of etoposide.     

Pharmacokinetics and First-pass Metabolism of Levomepromazine in the Rat

Abstract: The time course of the blood concentrations of levomepromazine and its two major non-polar metabolites in man were studied in the rat after single oral and intraarterial doses of levomepromazine hydrochloride. The blood levels of N-monodesmethyl levomepromazine were on average 179% of the levomepromazine levels after oral doses, but only 15% of the levomepromazine levels after intraarterial doses. The blood levels of levomepromazine sulfoxide, relative to the levomepromazine levels, were also generally higher after oral doses than after parenteral doses, on average 65% and 25%, respectively. Large interindividual variations were observed in the blood levels of levomepromazine and in the systemic availability of the drug after oral doses. The distribution phase lasted for about 8 hrs, the mean apparent volume of distribution was 16.6 l/kg, and the total body clearance was on average 12.3 ml/min. It is concluded that the rat provides a suitable model for the kinetics of levomepromazine in man, and that the sulfoxide and N-monodesmethyl metabolites of the drug are mainly formed by first-pass metabolism after oral doses in the rat, either in the liver or in the gut.     

液相色谱-串联质谱法研究白花前胡甲素脂质体在大鼠体内的药动学

目的制备白花前胡甲素脂质体并进一步研究其在大鼠体内的药动学。方法采用乙醇注入法制备白花前胡甲素脂质体,采用正交设计优化处方。白花前胡甲素脂质体按5、10、20mg·kg~(-1)低、中、高3个剂量经大鼠颈静脉给药后检测一定时间点的血药浓度,对照组为10mg·kg~(-1)的白花前胡甲素溶液。结果制备的脂质体含药量为2g·L~(-1),包封率为93.2%,脂质体高、中、低剂量组及对照组的t_(1/2)分别为(136.58±22.86),(74.12±6.97),(44.93±7.47),(51.26±5.13)min;AUC_(0-∞)分别为(215.93±33.24),(91.75±26.47),(29.22±4.47),(66.90±14.54)min·mg·L~(-1)。结论成功制备了白花前胡甲素脂质体,制得的白花前胡甲素脂质体在大鼠体内半衰期显著延长,生物利用度有明显的提高。     

The Effect of Amiodarone on Theophylline Pharmacokinetics in the Rat

Amiodarone is an investigational antiarrhythmic agent which has been implicated in reducing the activity of the hepatic mixed-function oxidase system. To evaluate this effect further, two groups of six male Sprague–Dawley rats each received theophylline (6 mg/kg, iv) preceded by either normal saline or amiodarone HC1 (100 mg/kg, iv). Blood samples were obtained serially for a period of 6 hr and the sera were assayed for theophylline by high-pressure liquid chromatography (HPLC). In rats pretreated with amiodarone, a significant 45% reduction in the mean (± SD) systemic clearance [0.057 (0.010) vs 0.031 (0.004) liter/hr/kg, P < 0.001] and a greater than 100% increase in the mean elimination half-life [2.03 (0.46) vs 4.29 (0.71) hr, P < 0.001] of theophylline were observed. These data demonstrate an acute inhibitory effect of amiodarone on the hepatic microsomal enzyme system.     

大鼠Smad 7基因真核表达质粒的构建与鉴定

目的：构建并鉴定大鼠Smad7/pcDNA3.1（＋）真核表达质粒,为进一步研究Smad7的抗肝纤维化作用提供实验基础。方法：TRIzol法抽提大鼠肝组织总RNA,琼脂糖凝胶电泳检测其完整性,紫外分光核酸蛋白分析仪测定其浓度与纯度,两步法获取大鼠Smad7cDNA片段,CaCl2法制备感受态细胞,应用EcoRI及XhoI在pcDNA3.1（＋）多克隆位点处进行双酶切,切胶回收载体及目的片段酶切产物,将回收的线性化pcDNA3.1（＋）与Smad7cDNA连接,构建Smad7/pcDNA3.1（＋）重组质粒,转化DH5α大肠杆菌,测序鉴定双酶切证实的阳性克隆。结果：电泳检测RNA的完整性良好,28S约为18S的2倍,A260/A280=1.986,纯度较好,阳性克隆酶切后电泳检测在DNAMarker1.3kb处见Smad7目的片段,5.4kb处见线性化pcDNA3.1（＋）,与预期结果相符,质粒重组成功,并测序证实。结论：大鼠Smad7真核表达质粒构建成功,为进一步单独或联合其他质粒从TGF-β/SMAD信号传导的不同环节干预肝纤维化提供了实验基础。     

Development of the Liver- and Lobe-Selective Nonviral Gene Transfer Following the Instillation of Naked Plasmid DNA Using Catheter on the Liver Surface in Mice

Purpose. The present study has undertaken the liver- and lobe-selective nonviral gene transfer following the instillation of naked plasmid DNA (pDNA) using catheter on the liver surface in mice. Methods. The polyethernylon catheter was inserted intraperitoneally through the abdominal wall and was retained on the surface of the liver right and left medial lobes. pDNA was administered through the catheter to the liver right and left medial lobes. Results. The luciferase levels produced in the applied liver lobes at 6 h after liver surface instillation of pDNA were significantly higher than those produced in other liver lobes and other tissues assayed, and ranged from approximately 5 folds higher in other lobes to 20-30 folds higher in other tissues. Following liver surface instillation of pDNA at a time from 2 to 24 h or at a volume from 15 to 60 l, the gene expressions of the applied liver lobes were always significantly higher than those of other liver lobes and other tissues. Conclusion. We have demonstrated the liver- and lobe-selective gene transfection following the instillation of naked pDNA using catheter on the liver surface in mice.     

Dose-Ranging Pharmacokinetics of Zidovudine (Azidothymidine) in the Rat

Pharmaceutical Research -     

Pharmacokinetics and tissue distribution profile of icariin propylene glycol-liposome intraperitoneal injection in mice

Objectives The pharmacokinetics and tissue distribution of icariin propylene glycol‐liposome suspension (ICA‐PG‐liposomes) have been investigated. Methods ICA‐PG‐liposomes or ICA‐PG‐solution were prepared and intraperitoneally injected to mice. Morphology and size distribution of ICA‐PG‐liposomes were observed by transmission electron microscopy (TEM) and laser particle sizer. Plasma and tissues were collected at different times after intraperitoneal injection and icariin concentrations were determined by HPLC. Key findings From TEM, ICA‐PG‐liposomes showed spherical vesicles with a mean particle size of 182.4 nm. The encapsulation efficiency of ICA‐PG‐liposomes reached 92.6%. Pharmacokinetics of ICA‐PG‐liposomes displayed the three open compartments model. ICA‐PG‐liposomes enhanced icariin absorption from the abdominal cavity, prolonged mean retention time (MRT_(0‐t)), increased area under curve (AUC_(0‐t)) and maximum concentration in plasma. Compared with ICA‐PG‐solution, ICA‐PG‐liposomes resulted in larger amounts of icariin being distributed into spleen (60.38% total icariin), liver (16.68%), lung (6.21%), kidney (4.64%), heart (1.43%) and brain (1.83%). AUC_(0‐t) values in most tissues (except lung) of mice administered ICA‐PG‐liposomes were higher than those administered ICA‐PG‐solution, while Clearance in most tissues (except brain and lung) decreased. The MRT_(0‐t) values of ICA‐PG‐liposomes in all tissues and half lives of most tissues (except brain) were prolonged. From Targeted efficiency and relative uptake data, the spleen was the target tissue of the ICA‐PG‐liposomes. Conclusions ICA‐PG‐liposomes changed the pharmacokinetic behaviour and enhanced icariin distribution in tissues. With nanometer size, high encapsulation efficiency and improved pharmacokinetics, ICA‐PG‐liposomes might be developed as promising carriers for icariin injection.     

落新妇苷固体分散体在大鼠体内的药动学研究

目的 研究大鼠口服落新妇苷固体分散体后的药物动力学参数。方法 采用HPLC法测定大鼠口服落新妇苷固体分散体后血浆中的落新妇苷血药浓度。用kinetica软件计算动力学参数。结果 落新妇苷在大鼠体内的药物动力学过程符合二室模型。AUC0-480min为1.98±0.60 mmol?min?L-1。结论 将落新妇苷制成固体分散体可以显著提高其生物利用度。     

Effect of Isoflurane Anesthesia on Antipyrine Pharmacokinetics in the Rat

Pharmaceutical Research -     

Significant Role of Liver Sinusoidal Endothelial Cells in Hepatic Uptake and Degradation of Naked Plasmid DNA After Intravenous Injection

Purpose. Uptake and degradation of naked plasmid DNA (pDNA) by liver sinusoidal endothelial cells (LSECs) were investigated. Methods. Tissue distribution and intrahepatic localization were determined after an intravenous injection of ¹¹¹In- or ³²P-labeled pDNA into rats. Cellular uptake and degradation of fluorescein- or ³²P-labeled pDNA were evaluated using primary cultures of rat LSECs. Results. Following intravenous injection, pDNA was rapidly eliminated from the circulation and taken up by the liver. Fractionation of liver-constituting cells by centrifugal elutriation revealed a major contribution of LSECs to the overall hepatic uptake of pDNA. Confocal microscopic study confirmed intracellular uptake of pDNA in cultured LSECs. Apparent cellular association of pDNA was similar at 37°C and 4°C. However, trichloroacetic acid (TCA) precipitation experiments showed the TCA-soluble radioactivity in the culture medium increased in an accumulative manner at 37°C. Involvement of a specific mechanism was demonstrated, as the uptake of pDNA was significantly inhibited by excess unlabeled pDNA and some polyanions (polyinosinic acid, dextran sulfate, heparin) but not by others (polycytidylic acid, dextran). These inhibitors also reduced the amount of TCA-soluble radioactivity in the culture medium. Conclusion. These results suggest that LSECs efficiently ingested and rapidly degraded naked pDNA in vivo and in vitro and released the degradation products into the extracellular space.