自然水体生物膜胞外蛋白质吸附铅和镉的研究

本文通过长春市南湖水中生物膜优势菌种胞外蛋白吸附Pb2+和Cd2+的实验, 研究了胞外蛋白吸附重金属的规律.     

两种新型99mTcN核标记的甲硝唑类乏氧显像剂的制备及生物性能评价

为研制新的肿瘤乏氧显像剂, 设计合成了2-(2-甲基-5-硝基咪唑基)乙基氨荒酸钾(MNIE-DTC)和4-(2-甲基-5-硝基咪唑基)丁基氨荒酸钾(MNIB-DTC)两种氨荒酸盐配体, 并制得了相应的99mTcN核配合物99mTcN(MNIE-DTC)2和99mTcN(MNIB-DTC)2. 所获得的两种99mTcN核配合物均为电中性, 具有较高的体外稳定性. 在荷乳腺癌的TA-2小鼠体内分布实验结果显示, 两种配合物均具有一定的肿瘤摄取, 给药1 h后, 99mTcN(MNIE-DTC)2和99mTcN(MNIB-DTC)2的肿瘤摄取率分别为(0.50±0.01)%ID/g和(0.64±0.10)%ID/g. 注入肼苯哒嗪后, 两种配合物的肿瘤摄取明显增高, 表明这两种配合物都具有对乏氧肿瘤的选择性.     

131I标记多肽YP13聚乙二醇修饰物及其生物分布

利用噬菌体展示技术, 对荷人结肠癌CL-187裸鼠进行体内筛选, 获得在肿瘤组织中富集的一条十二肽SVSVGMLPSHAP. 为了标记131I, 在N端连接酪氨酸合成了十三肽YP13(YSVSVGMLPSHAP). 另外, 利用单甲基化聚乙二醇(mPEG5000)对YP13进行化学修饰. RP-HPLC分离纯化YP13、mPEG-YP13和对照随机十三肽YR13(YEDIKPKTSLAFR) 131I标记产物, 放化纯度大于95%. RP-HPLC分析131I-YP13和131I-mPEG-YP13体内循环60 min后的血清, 结果显示, 131I-mPEG-YP13体内稳定性优于131I-YP13. 这三种标记物在荷人结肠癌CL-187裸鼠体内的分布表明, 131I-YP13和131I-mPEG-YP13在1和2 h时的肿瘤摄取远远高于对照肽131I-YR13, 二者的瘤血比随时间的延长而升高. 131I-mPEG-YP13在肿瘤中的摄取随着时间的延长有所改善. 因此, 放射性碘标记的多肽YP13及其聚乙二醇修饰物可能成为新型结肠癌显像剂.     

新的99mTc标记σ受体肿瘤显像剂

采用整体法设计合成了新的^99mTc标记的配合物[N-[2-((2-oxo-2-(4-(3-phenylpropyl) piperazin-1-yl)ethyl)(2-mercaptoethyl)amino)acetyl]-2-aminoethanethiolato]technetium(Ⅴ) oxide (PPPE-MAMA′-^99mTcO)([^99mTc]-2)及其相应的铼配合物(PPPE-MAMA′-ReO)(Re-2). 竞争结合实验表明Re-2对σ₁和σ₂受体有中等亲和力, K_i值分别为8.67 ± 0.07和5.71 ± 1.88 μmol/L; 荷MCF-7人乳癌裸鼠尾静脉注射[^99mTc]-2后0.5 h, 4 h, 20 h采集平面图像, 20 h时可以看到肿瘤部位有放射性浓集, 共同注射[^99mTc]-2和抑制剂氟哌啶醇(1 mg/kg)后显像, 20 h时肿瘤部位无明显放射性浓集; 体内生物分布结果显示, 注射后24 h肿瘤中的放射性摄取为0.14% ± 0.01% ID/g, 肿瘤/肌肉比为6.02 ± 0.87. 上述结果表明: 虽然用整体设计法对前体化合物的结构进行了较大修饰, 但得到的^99mTc-配合物([^99mTc]-2)在肿瘤内仍有一定的浓集, 与σ₁和σ₂受体仍保持一定的亲和力. 在此配合物的基础上, 对其进行进一步的结构修饰有可能得到对σ受体亲和力更高的肿瘤显像剂.     

镧对保卫细胞质膜内向钾通道的作用研究

用膜片钳全细胞记录方式研究了La³⁺对蚕豆气孔保卫细胞质膜内向钾通道(K⁺_in)电流的影响. 结果表明, 在细胞外液和细胞内液中的La³⁺对K⁺_in电流都产生抑制作用, 说明在保卫细胞质膜K⁺_in通道的细胞膜外侧和内侧存在La³⁺的结合位点; 在细胞外La³⁺对K⁺_in电流的阻断常数K_i为(2.56±0.25) mmol/L, 在细胞内La³⁺对K⁺_in电流的阻断常数K_i为(1.18±0.11)×10^−15 mol/L, 可见La³⁺在细胞内比细胞外对K⁺_in电流产生更强的抑制作用, 也说明在蚕豆保卫细胞质膜K⁺_in通道, 细胞膜内侧比外侧存在更强的La³⁺结合位点. 离子通道的活性变化与植物叶片上的气孔运动和植物水分状况密切相关, 这提示稀土对于调节植物的水分散失, 增强植物耐旱性可能具有潜在的应用价值.     

卤化银添加剂对125I在北山花岗岩岩粉中的吸附作用研究

利用柱法实验和批次实验研究了卤化银(AgCl、AgBr、AgI)对¹²⁵I在甘肃北山花岗岩中的吸附现象。结果表明在无载体的情况下,卤化银添加剂有利于¹²⁵I在北山花岗岩岩粉中的吸附,有效分配系数(K_d,e)均大于1.0×10³ mL·g^-1。在有载体的情况下,氯化银和溴化银仍然对¹²⁵I在北山花岗岩岩粉中有良好的吸附作用,K_d,e大于1.0×10³ mL·g^-1。其中,溴化银的吸附效果最好,这可能与其晶体结构和光解性质有关。     

Na+/Ca2+交换调节的La3+跨淋巴细胞膜的定量研究

利用荧光浓度指示剂fura-2研究了La³⁺能否利用Na⁺/Ca²⁺交换系统进入人外周血淋巴细胞以及La³⁺Na⁺/Ca²⁺交换的影响. 并首次用此方法研究了La³⁺能否在细胞器(主要为内质网和线粒体)中蓄积. 实验结果表明, 用乌本苷预处理细胞并在无Na⁺介质中测试, 可明显观察到La³⁺跨膜进入淋巴细胞, 而且胞内La³⁺的浓度与胞外的La³⁺浓度成正比. 但当胞外La³⁺浓度大于0.4 mmol/L时, 不再观察到340/380 nm荧光比值的变化, 此时细胞内La³⁺浓度约为1.5×10^-12mol/L. 当La³⁺浓度较大时(0.1 mmol/L)抑制Na⁺/Ca²⁺交换操纵的Ca²⁺的进入, 而较低浓度(0.01 mmol/L)时却刺激Ca²⁺进入. 另外从实验结果可推测La³⁺可以被依赖ATP的质膜钙泵泵出胞外. 胞内钙库用离子霉素耗竭后, La³⁺内流过程中再次加入离子霉素后, 可明显观察到340/380 nm荧光比值增大, 说明La³⁺在细胞器中有一定程度的蓄积. 在模拟胞内离子组分的缓冲液中(pH = 7.05), fura-2对La³⁺的检测限为10^-12mol/L, 对Ca²⁺的检测限为10^-8 mol/L, 并测得fura-2-La³⁺的络合比为1∶1, 表观离解常数为1.7×10^-12 mol/L.     

朱砂中汞的生物可接受性及其吸收与排泄

研究了朱砂中汞的生物可接受性及其在体内的吸收与排泄.采用体外消化透析法测定了朱砂中汞的生物可接受性;计算了大鼠灌胃给予临床剂量(50mg/kg)朱砂后汞的药动学参数;测定了给予临床剂量的朱砂后大鼠粪样中汞的排泄量.结果表明,朱砂中汞的溶出率为0.011%,生物可接受率为0.003 3%.大鼠灌胃给予临床剂量的朱砂后,汞的药动学参数为:最高血药浓度(ρmax)为(6.3±1.3)μg/L,达峰时间(tmax)为(1.3±0.4)h,半衰期(t1/2)为(4.2±0.5)h,血药浓度-时间曲线下面积(AUC)为(54.7±8.7)μg.h.L-1.给予朱砂12h后汞在粪便中排泄量最大,96h后在粪样中仍可检测到少量汞.朱砂中汞的生物可接受性较低,在体内吸收少,滞留时间较长,排泄缓慢,长期服用可在体内蓄积,产生毒性.     

人宫颈癌细胞(Hela)内的31P核磁共振研究

建立了无损伤性³¹P NMR研究细胞内物质的实验方法, 并对人宫颈癌细胞(Hela)的³¹P NMR谱中含磷小分子代谢物的谱峰进行了分析; 细胞内无机磷(Pi)的化学位移对pH非常敏感, 通过测定其化学位移可间接确定细胞内的pH, Hela细胞内Pi峰的化学位移为5.88±0.01 (n＝3), 计算得到细胞内 pH值为7.05±0.01; 通过测量Hela细胞的³¹P NMR谱中ATP的α磷和β磷及γ磷的化学位移差值, 得出Hela细胞内Mg^2＋与ATP结合的复合物MgATP和整个ATP量的比值, 计算得到Hela细胞内游离Mg^2＋浓度为(253.3±0.13) mmol/L (n＝3), 与其它分析方法相比, ³¹P NMR测定细胞内游离Mg^2＋浓度具有对细胞样品无损伤的优点.     

蛋白质与几个天然抗癌药物间相互作用的毛细管电泳研究

药物进入人体后,总要通过血浆的贮存和运输,达到受体部位后才发生药理作用.多数药物在血浆中都或多或少地会与血浆蛋白(主要是白蛋白)结合,结合后的药物不易穿透毛细管壁,限制了进一步运输.同时,药物与白蛋白结合后将存留于血浆中,减弱了药物的最大作用强度,延长了药物的作用时间~([1,2]).因此,药物与蛋白质的结合作用对药物在体内的代谢和分布有重要影响.     

Pharmacokinetic and brain distribution study of an anti-glioblastoma agent in mice by HPLC–MS/MS

Previously compound I showed great anti-glioblastoma activity without toxicity in a mouse xenograft study. In this study, a sensitive and rapid high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to investigate the pharmacokinetics and brain distribution of compound I in mice. The protein precipitation method was applied to extract the compound from mouse plasma and brain homogenates, and it was then separated using a Kinetex C₁₈ column with a mobile phase consisting of acetonitrile–0.1% formic acid water (50:50, v/v). The analytes were detected with multiple reaction monitoring for the quantitative response of the compounds. The inter- and intra-day precisions were <8.29 and 3.85%, respectively, and the accuracy range was within ±7.33%. The method was successfully applied to evaluate the pharmacokinetics of compound I in mouse plasma and brain tissue. The peak concentration in plasma was achieved within 1 h. The apparent elimination half-life was 4.06 h. The peak concentration of compound I in brain tissue was 0.88 μg/g. The results indicated that compound I was rapidly distributed and could cross the blood–brain barrier. The pharmacokinetic profile summarized provides valuable information for the further investigation of compound I as a potential anti-glioblastoma agent.     

HPLC Analysis and Pharmacokinetics of Picroside I in Dog Plasma

A sensitive and selective HPLC–UV method established for determination of picroside I in dog plasma has been used to study the pharmacokinetics of the drug after intravenous administration of three different doses. Sample pretreatment consists in deproteination by addition of acetonitrile; l-ascorbic acid was used to improve the stability of picroside I. The lower limit of quantification of picroside I was 0.05 μg mL⁻¹. The recovery of the method was up to 90%. After intravenous administration to dogs picroside I was mainly distributed in the central compartment and was rapidly eliminated from the plasma; the mean elimination half-life was 30.54 ± 4.34, 30.20 ± 3.78, and 34.02 ± 1.88 min for doses of 2.5, 5, and 15 mg kg⁻¹, respectively, and the respective values of AUC _0–∞ were 81.04 ± 19.95, 198.50 ± 27.77, and 586.44 ± 103.08 μg min mL⁻¹. The different doses had no significant effect on the main pharmacokinetic data and the kinetics seemed to be linear in dosage range 2.5–15 mg kg⁻¹.     

Study of the protein-bound fraction of calcium,iron, magnesium and zinc in bovine milk

Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry (ICPOES). Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g l⁻¹ trichloroacetic acid (TCA), and an enzymatic protein hydrolysis using 50 g l⁻¹ pepsin (PEP) solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions.     

A quantitative LC/MSMS method for determination of edoxaban,a Xa inhibitor and its pharmacokinetic application in patients after total knee arthroplasty

Edoxaban was extracted from human plasma by simple protein precipitation with acetonitrile, followed by quantitative determination using a liquid chromatography–mass spectrometry method. The recoveries of edoxaban and the internal standard (ticlopidine) from human plasma were >85%, and the within‐ and between‐day coefficients of variation were within 15%. The limit of quantification in human plasma was 1 ng/mL. The concentration of edoxaban in blood decreased at room temperature, but remained unchanged for 1 week at 4°C. On the other hand, the concentration in plasma at both −20 and −80°C remained unchanged for 5 months. These results indicated that blood samples should be centrifuged immediately or stored at 4°C, and that plasma samples should be stored below −20°C until analysis. This method was applied to human plasma obtained from four patients after total knee arthroplasty. Analysis of edoxaban pharmacokinetics demonstrated an absorption time lag of 4h, a maximum concentration of 110 ± 26 ng/mL and an oral clearance of 37 ± 16 L/h. The analytical methods established in this study will be suitable for determining the concentrations of edoxaban in human plasma.     

Applications of high throughput microsomal stability assay in drug discovery

High throughput in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with high throughput assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between high throughput in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds in order to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. In this study the relationship between in vitro single time point high throughput microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies was examined using 306 real world drug discovery compounds. The results showed that in vitro Phase I microsomal stability t(1/2) is significantly correlated to in vivo clearance with a p-value<0.001. For compounds with low in vitro rat microsomal stability (t(1/2)<15 min), 87% showed high clearance in vivo (CL>25 mL/min/kg). This demonstrates that high throughput microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability (t(1/2)>15 min), no significant differentiation was observed between high and low clearance compounds. This is likely owing to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance. This finding supports the strategy used by medicinal chemists and drug discovery teams of applying the in vitro data to triage compounds for in vivo PK and efficacy studies and guide structural modification to improve metabolic stability. When in vitro and in vivo data are both available for a compound, potential in vivo clearance pathways can be diagnosed to guide further discovery studies.     

Determination of curcumol in rat plasma by capillary gas chromatography with a hydrogen flame ionization detector

A simple and sensitive capillary gas chromatography with a hydrogen flame ionization detector (GC‐FID) method was developed for the determination of curcumol in rat plasma. From a variety of compounds and solvents tested, buagafuran was selected as the internal standard (IS) and acetonitrile was found to be the best protein precipitation agent and solvent for extracting curcumol from plasma and tissues samples. (Buagafuran was used as an internal standard. Curcumol was extracted by a protein precipitation with acetonitrile.) The samples were determined by GC on an HP‐5 column (30.0 m × 0.32 mm, 0.25 μm); inlet volume 2 μL; split ratio 10 : 1; inlet temperature 250°C; oven temperature 180°C; flow 1.0 mL/·min; FID 250°C; carrier gas N₂. The resulting retention times of curcumol and IS were 6.0 and 9.5 min. There was good linearity over the range 0.133–133.3 μg/mL (r = 0.9999) in plasma samples. The method recoveries were 97.7–102.0% in plasma, and the intra‐ and inter‐day variances (RSD) were less than 15% in all cases. The GC method was applied to develop a pharmacokinetics study in which experimental rats received a single administration of curcumol by intravenous injection. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis

This paper investigates the preparation of Fasciola hepatica samples for two-dimensional electrophoresis (2-DE). Whole samples were prepared by both hot sodium dodecyl sulfate (SDS) solubilisation and precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants and to inactivate endogenous proteases. Sample preparation had a marked influence on the 2-DE gel profile. TCA precipitation resulted in no measurable improvement in the profile observed, compared to the untreated control. Solubilisation of sample with hot SDS increased the number of protein spots, as did TCA precipitation with the addition of phosphotungstic acid. The preparation of excretory-secretory (ES) products poses problems due to both high salt concentrations and low protein concentration. All precipitation methods used to overcome this gave similar profiles, except acetone alone, which caused depletion of the larger proteins. TCA in acetone gave the best result, similar to that obtained by centrifugal filtration of the sample. Overcrowding of spots in some regions of the 2-DE gel occurred in the whole Fasciola hepatica sample. This problem was alleviated by differential solubilisation, which also resulted in the enrichment of some proteins.     

In vitro evaluation of radiolabeled (125I) methanol extracts of yarrow in cell lines of MCF-7, PC-3, A-549 and Caco-2

Nowadays, cancer is still the second leading cause of death all over the world. Therefore, natural products which have anticancer and antitumor properties are come into prominence. Achillea family is known with anticancer and antitumor activity. Yarrow which has over a hundred bioactive compounds is a member of Achillea family. In current study; components of yarrow which was obtained after methanol extraction and purification were radiolabeled with ¹²⁵I and effects of these radiolabeled components on the cells were examined with using Caco-2, MCF-7, A-549, PC-3 cell lines. As a result of these studies, seven peaks were obtained and the highest radiolabeling yield was calculated for ¹²⁵I radiolabeled Peak 7 (95.00 ± 7.07, n = 4). To screen the biological properties of these radiolabeled peaks at determined cell lines, our ongoing effort was to evaluate incorporation percentage with time dependent. Furthermore, ¹²⁵I-Peak 7 had highest incorporation ratio for whole cell lines and its incorporation percentage was increased with time dependent. Results of these in vitro studies were compatible with previous in vivo studies and traditional use of yarrow plants.     

The Application of Trichloroacetic Acid as an Ion Pairing Reagent in LC–MS–MS Method Development for Highly Polar Aminoglycoside Compounds

A simple, sensitive and robust liquid chromatography-tandem mass spectrometry method was developed and validated for highly polar aminoglycoside compounds gentamicin, kanamycin and apramycin. The effect of trichloroacetic acid (TCA) concentration on plasma protein precipitation and sample recovery was studied and an optimized concentration of 25–30% TCA were determined that gives the best sample recovery for aminoglycosides from rat plasma. The effect of TCA concentration on the chromatographic behavior of gentamicin and tobramycin was studied on a Synergy Max RP-column using a mobile phase with a pH of 2.78. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 1–5,000, 20–10,000, 10–10,000 ng mL^?1 with precisions of 2.6–4.1, 3.3–5.0, 1.5–9.9%, and accuracies of 94.7–103.7, 87.9–104.9, 91.3–103.6% for gentamicin, kanamycin and apramycin, respectively. The LLOQs corresponding with a coefficient of variation less than 20% were 1, 20 and 10 ng mL^?1 for gentamicin, kanamycin and apramycin, respectively.     

Radioiodinated paroxetine, a novel potential radiopharmaceutical for lung perfusion scan

Paroxetine (a selective serotonin reuptake inhibitor) was successfully labeled with ¹²⁵I via direct electrophilic substitution reaction at ambient temperature. The reaction parameters studied were paroxetine amount, CAT amount, pH of the reaction mixture, reaction temperature, reaction time and in vitro stability of ¹²⁵I-paroxetine. ¹²⁵I-paroxetine was obtained with a maximum labeling yield of 94 ± 0.23% and in vitro stability up to 24 h. Biodistribution studies showed that maximum in vivo uptake of ¹²⁵I-paroxetine in lungs was 27.89 ± 1.03% injected activity/g tissue at 15 min post-injection and retention in lungs remained high up to 1 h, whereas the clearance from mice appeared to proceed mainly via the hepatobiliary pathway. ¹²⁵I-paroxetine is not a blood product and so it is more safe than the currently available ^99mTc-macroaggregated albumin (^99mTc-MAA), and its lung uptake is higher than that of the recently discovered ^99mTc(CO)₅I and ^99mTc-DHPM. As a conclusion, radioiodinated paroxetine could be used as a novel radiopharmaceutical for lung perfusion scan safer than the currently available ^99mTc-MAA and more potential than the recently discovered ^99mTc(CO)₅I and ^99mTc-DHPM.