Table of Content

15 June 2008, Volume 17 Issue 2

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Journal of Chinese Pharmaceutical Sciences

2008, 17(2):  93-96. 

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In vitro investigation of RGD-modified stabilized cationic liposomes as non-viral vehicle for siRNA delivery

Shi-Jin Yang, Li-Juan Yang, Juan Jiang, Jian-Cheng Wang^*, Qiang Zhang

2008, 17(2):  97-105. 

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In this study, the novel RGD-modified stabilized cationic liposomes were developed as the delivery vehicle for siRNA targeting human MDR1 gene. The complex of cationic liposomes and siRNA, RGD-Lipo-siRNA, was prepared with a narrow size distribution below 200 nm. It was shown that the encapsulated siRNA in the liposomes could be effectively protected from serum degradation. Also, enhanced cell binding and intracellular uptake of siRNA in the doxorubicin-resistant human ovarian cancer cell lines SKOV3/A were found in RGD-Lipo-siRNA preparation as compared to that of unmodified cationic lipsomes (Lipo-siRNA). Using the post-insertion method for RGD modification, lysosome release of siRNA in pRGD-Lipo-siRNA was improved. From flow cytometry, significant increase of doxorubicin accumulation was observed in the SKOV3/A cells treated with pRGD-Lipo-siRNA targeting human MDR1 gene. In vitro cytotoxicity assay showed that the significant cell growth inhibition was achieved in the SKOV3/A cells after treating with the combined use of siRNA and doxorubicin. In conclusions, postinserted RGD modified lipoplex, pRGD-Lipo-siRNA, was successfully used for siRNA transfection and achieved drug resistance reversal in human ovarian cancer SKOV3/A (doxorubicin-resistant) cells. It suggested that this liposomes might be a potential vehicle for siRNA delivery in vivo.

Tissue distribution, excretion and pharmacokinetics of R-hap in rats

Jia-Yu Zhou, Zhi-Yun Meng, Gui-Fang Dou*, Shou-Ting Fu, Zi-Chun Hua,
Xiang Yang, Jun Liu, Li-Jun Wei, Sun-Hua Zhang

2008, 17(2):  106-112. 

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The purpose of this study is to characterize the tissue distribution, excretion and pharmacokinetics profiles of R-hap in healthy Wistar rats. R-hap was radiolabeled by the IODO-GEN method. Tissue distribution and urinary, fecal and biliary excretion patterns of 125I-R-hap were investigated following a single i.v. bolus injection. Pharmacokinetics properties of 125I-R-hap were also examined after a single i.v. bolus injection. The trichloroacetic acid (TCA) precipitated radioactivity was widely distributed and rapidly diminished in most tissues. Kidney contained the highest radioactivity among all organs and the distribution of 125I-R-hap to fat was minimal. The cumulative excretion of 125I-R-hap reached 71.81% ± 2.15% of the administered radioactivity at 48 h and 94.71% ± 1.50% at 120 h. Urinary excretion was the dominant route of elimination following i.v. administration, as 80.64% ± 1.47% and 14.07% ± 0.95% of administered radioactivity were recovered in urine and feces, sectively, in intact rats over 120 h. The mean areas under the plasma concentration-time curve was (8818.4 ± 576.1) Bq/h/mL. The results of tissue distribution, excretion and pharmacokinetics of R-hap in rats provided biopharmaceutical basis for the design of future clinical trials.

Preparation, physicochemical characterization and cyctotoxicity of solid
dispersion of paclitaxel and polyvinylpyrrolidone

Jia-Bei Sun, Xiang-Rui Liu, Jian-Cheng Wang, Wan-Liang Lu, Xuan Zhang*,
Qiang Zhang

2008, 17(2):  113-117. 

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The objective of this study was to prepare and characterize paclitaxel-polyvinylpyrrolidone (PTX-PVP) solid dispersions with the intention of improving its solubility and dissolution properties. The PTX-PVP solid dispersion systems were prepared by solvent method. The release rate of paclitaxel was determined from dissolution studies and the physicochemical properties of solid dispersion were investigated by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM). The cytotoxicities of paclitaxel in solid dispersion to the SKOV-3 cells were assayed by a SRB staining method. The results showed that the solubility and dissolution rate of paclitaxel were significantly improved in solid dispersion system compared with that of the pure drug and physical mixture. The results of DSC and PXRD showed that the paclitaxel in solid dispersion was amorphous form. No paclitaxel crystals in the solid dispersions was found during SEM analysis. Cytotoxicity study suggested that the inhibitory rates of PTX-PVP solid dispersion to SKOV-3 cells were higher than that of pure paclitaxel. The solubility and dissolution of paclitaxel were improved by solid dispersion technique. In vitro cytotoxicity of paclitaxel in solid dispersion was higher than that of pure drug.

Pharmacokinetics of recombinant human parathyroid hormone after subcutaneous administration in Rhesus monkeys by immunoradiometric assay

Xue-Wei Song, Zhi-Hang Chen, Jin-Jing Che, Cheng-Qi Shan, Yu-Nan Hou,
Ren-Jiu Zheng, Yuan-Guo Cheng*

2008, 17(2):  118-121. 

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The purpose of this research was to study the pharmacokinetics and the bioavailability of recombinant human parathyroid hormone [rhPTH (1-34)] in Rhesus monkeys after single and multiple subcutaneous administration. An immunoradiometric assay (IRMA) was used to determine the plasma drug concentration of rhPTH (1-34) after giving single dose of 10, 20 and 40 μg/kg and daily dose of 40 μg/kg for 7 d by subcutaneous administration, and intravenous injection of 20 μg/kg in Rhesus monkeys. The pharmacokinetic parameters were calculated by noncompartmental analysis. The drug plasma level quantitation range was from 0.027 to 2.22 ng/mL. The intra- and inter-assay precision (CV) of analysis were less than 15%, and the average recovery was about 93.0% ± 8.6% - 116.5% ± 14.0%. After subcutaneous administration of rhPTH(1-34) at dose of 10, 20 and 40 μg/kg, the average Tmax was 0.67, 0.5 and 0.83 h, Cmax were 1.85 ± 0.05, 3.23 ± 0.25 and 7.15 ± 1.19 ng/mL, the AUC(0-∞) were 3.4 ± 0.6, 10.7 ± 1.3 and 12.6 ± 1.5 ng/h/mL, and terminal-phase elimination T1/2 were 0.72 ± 0.10, 1.15 ± 0.10 and 1.03 ± 0.06 h, respectively. The absolute bioavailability of rhPTH (1-34) was 46.96% after subcutaneous administration of 20 μg/kg. There was no evidence of accumulation during systemic exposure of rhPTH (1-34) upon multiple dosing in Rhesus monkeys. The IRMA assay method provide reasonable sensitivity and specificity for the pharmacokinetic study of rhPTH (1-34) after subcutaneous or intravenous administration in Rhesus monkeys. The pharmacokinetic characteristic of rhPTH (1-34) in monkeys shows linear relationship with the dose administered subcutaneously.

Determination of penehyclidine hydrochloride in beagle dog plasma by liquid chromatography-electrospray ionization mass spectrometry and the pharmacokinetic study

Yan Cui, Hai-Lin Yin, Xu Bao, Mei-Jin Xiong, Cong Chen, Li-Ming Ye*

2008, 17(2):  122-128. 

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To develop a fast and sensitive liquid chromatography-mass spectrometry method for the determination of penehyclidine hydrochloride (PH) in beagle dog plasma. PH and diphenhydramine hydrochloride (internal standard, IS) were extracted with a solvent mixture of petroleum ether-ethyl ether (7:3). Chromatographic separation was achieved on a reversed-phase Eclipse XDB-C18 column (4.6 mm×150 mm, 5 μm) using the eluent of methanol-water (5 mmol/L ammonium acetate) (90:10, v/v, pH 5.8) as mobile phase. The electrospray ionization source was set at the positive multiple reaction monitoring (MRM) mode. This method involved the use of the [M+H]⁺ ions of PH and diphenhydramine hydrochloride at m/z 316.4-128.2 and m/z 256.4-167.2. The calibration curve was linear in the range of 1-1000 ng/mL with a correlation coefficient of 0.9988. The lower limit of quantification was 0.05 ng/mL. The precision, accuracy and recovery of the method were acceptable. Following intravenous injection administration at doses of 0.5, 1 and 5 mg/kg PH, the main pharmacokinetic parameters were as the followings, t1/2α 0.33 h, t1/2β 2.44 h, tmax 0.058 h, AUC and Cmax exhibited a linear increase along with the increase of dose. The two-compartment model fit the three dose groups. This method was sensitive, accurate and fast for the determination of concentration of PH in beagle dog plasma. It could be used in pharmacokinetic studies of PH.

Synthesis and biological evaluation of neamine analogues for RNA binding

Li Cai, Bo Ren, Feng-Rong Zhang, Zhen-Jun Yang*, Liang-Ren Zhang,
Li-He Zhang

2008, 17(2):  129-133. 

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To design and synthesize neamine analogues modified at 5 position of ring II, which could improve the binding affinity of aminoglycosides to 16S RNA. Started from neomycin B, modified neamine analogues were synthesized through organic reactions such as hydrolysis, protection, nucleophilic substitution, deprotection and reduction. The interaction of the target compounds with A-site RNA in E. coli. ribosome (16S RNA) was determined by surface plasmon resonance (SPR), respectively. Six target compounds were synthesized. Some of them showed antibacterial activities and enhanced affinity to 16S RNA at 10–3 M in vitro. Introduction amino or aliphatic amino group at 5 position of ring II in neamine would maintained antibacterial activities as well as increase binding affinity to 16S RNA. Furthermore, there is almost no influence on the stability of drug/16S RNA complex by inverting the configuration of 5-hydroxyl group at ring II.

Improved synthesis and characterization of L-histidine norcantharimide,
a novel potent protein phosphatase 2A inhibitor

Da-Feng Chen, Yong Zou*, Yong-Qiang Li, Yu-Chen Cai, Li-Jian Xian

2008, 17(2):  134-137. 

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Improved synthesis and structure identification of L-histidine norcantharimide, a potent PP2A inhibitor was reported. Condensation between norcantharidin and L-histidine in 95% EtOH at reflux temperature affords L-histidine norcantharimide in 97.0% yield which is much higher compared with literature, and more importantly, the configuration is retained. The chemical structure of the compound was re-elucidated through IR, FAB-MS, ¹H NMR, ¹³C NMR and 2D NMR (¹H, ¹³C-COSY and HMBC), the fundamental physical data, including optical data being also firstly reported. Preliminary cytotoxicity evaluation showed that the target compound was probably more potent than norcantharidin against a panel of human cancer cell lines. Design and synthesis of amino acid (nor) cantharimides would provide a convenient and rational structure modification of (nor) cantharidin and open new avenues to explore new promising candidates.

Synthesis of kanamycin A derivatives by regioselective masking drug resistant enzymes targeting hydroxyl groups

Ying Chen, Xiang-Bao Meng, Gui-Hui Chen, Pan Pan, Zhong-Jun Li^*

2008, 17(2):  138-143. 

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The 3'-OH, 4'-OH and 2''-OH of kanamycin A were modified in search of new aminoglycosides to overcome resistant enzymes, ANTs and APHs. The key intermediate was a dibenzylidene-protected derivative of kanamycin A. The aimed sites were masked by benzyl, methyl and allyl groups. Multi-step reactions gave the desired aminoglycoside derivatives but showed less antibiotic activity than kanamycin A.

Chemical constituents from the aerial parts of Plumbago zeylanica L.

Xiao-Yan Huang, Ming-Xiong Tan, Qiang Wu, Yong Chen, Heng-Shan Wang^*

2008, 17(2):  144-147. 

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To investigate the chemical constituents from the aerial parts of Plumbago zeylanica L. The chemical constituents were isolated by various column chromatographic methods and the structures were elucidated by various spectroscopic methods, especially 2D NMR spectra. A new triterpenoid, 1β,3β,11α-trihydroxy-urs-12-ene (1), together with six known compounds, androsta-1,4-diene-3,17- dione (2), isoshinznolone (3), neoechinulin A (4), harman (5), ergostadiene-3β,5α,6β-triol (6) and N-(N'-benzoyl-S-phenylalaninyl)-Sphenylalaninol (7) were isolated from the aerial parts of P. zeylanica. Compound 1 was a new compound, and compounds 2, 4-7 were obtained from this genus for the first time.

Chemical constituents from the roots of Polygala sibirica L.

Yu-Hong Zhou, Yong Jiang, Jing Wen, Yu-Ping Chen, Peng-Fei Tu*

2008, 17(2):  148-152. 

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To investigate the chemical constituents of the roots of Polygala sibirica L. The separation and purification were performed by solvent extraction and repeated chromatography with silica gel, Sephadex LH-20, ODS columns, and semiprep. HPLC. The structures were elucidated by spectral analysis. Twelve known compounds were isolated and identified as tenuifoliside A (1), tenuifoliside B (2), glomeratose A (3), 3',6-disinapoyl sucrose (4), sibiricose A5 (5), sibiricose A6 (6), sibiricose A1 (7), sibiricose A2 (8), polygalatenoside E (9), 1-O-L-arabinopyranosyl-O-(6→1)-β-D-glucopyranosyl-salicylate (10), canthoside A (11), and methyl- 3,4,5-trimethoxycinnamate (12). Compound 11 was obtained from genus Polygala for the first time, and compounds 2, 9, 10 and 12 were isolated from this plant for the first time.

Purification method of rohitukine from the stem bark of Dysoxylum binectariferum

Bao-Song Cui, Xiao-Qing Ma, Dong-Hui Yang*, Xue-Qiao Hu, Shao-Qing Cai

2008, 17(2):  153-157. 

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To develop a simple and rapid purification method of rohitukine from the stem bark of Dysoxylum binectariferum. A L9(3⁴) orthogonal test was designed to optimize the extraction condition. Rohitukine in the plant extract was purified by using solvent-solvent partition and cation exchange resion (CER). Five different types of packing materials, including XAD-2 resin, polyamide, Sephadex LH-20, ODS and CER, were compared and CER showed the best capacity for rohitukine separation. The purification procedure was optimized as follows: the plant material powder was extracted with 70% ethanol (v/m = 60) by ultrasonic agitation for 60 min, then the 70% ethanol extract was dissolved in aqueous solution (pH 1, adjusted with 0.5 mol/L HCl) and extracted with equal volume of n-butanol. The aqueous layer was retained and the pH was adjusted to 10 with 25% aqueous ammonia and a solventsolvent partition was performed with equal volume of n-butanol. The obtained n-butanol extract was dissolved in aqueous solution (pH 1, adjusted with 0.5 mol/L HCl), and purified by a CER column eluting with H2O and 70% ethanol (pH 10, adjusted with 25% aqueous ammonia), successively. Rohitukine existed in 70% ethanol eluate, with a purity up to 53.3%. The method developed in this study provides a simple and rapid approach for the preparation of rohitukine from the stem bark of D. binectariferum.

Determination of trans-resveratrol in mouse liver by high performance liquid chromatography

Qian Yao, Shi-Xiang Hou, Xi-Hui He, Xuan Zhang, Jun Yan, Xiao-Jun Gou^*

2008, 17(2):  158-162. 

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To develop a sensitive high performance liquid chromatography (HPLC) assay for the determination of trans-resveratrol in mouse liver. The whole liver of a mouse was removed from the body, homogenated, and extracted by ethyl acetate. The organic layer was isolated and evaporated to dryness, the residue was reconstituted in 0.2 mL mobile phase for centrifugation, and 50 μL of the supernatant was injected into the HPLC instrument. The sample was separated on a Shimadzu ODS column (150 mm × 4.6 mm, 5 μm) at 35 °C and detected by ultraviolet (UV) detector at the wavelength of 305 nm. The mobile phase consisted of methanol and 0.1 mol/L acetic acid (4:6, v/v) with the flow-rate at 1 mL/min. The limit of detection was 3.0 ng/g in liver homogenate with a signal/noise ratio of 3:1. The linear range of the calibration curve was 5.0-120.0 ng/g. The mean recoveries at the concentrations of 6, 10 and 80 ng/g were 102%, 96.0% and 91.5%, respectively. The RSDs for inter- and intra-day assays were less than 5%. Compared with other reported methods, this method was faster and more sensitive. It was also proved to be of good linearity, selectivity, accuracy and precision, and can be efficiently applied to the pharmacokinetic study of trans-resveratrol in mouse liver.

Determination of magnolol and honokiol in traditional Chinese medicine
Magnolia officinalis and its preparations by liquid-phase microextractionback
extraction combined with high performance liquid chromatography

Xiao-Yuan Wang, Xuan Chen, Hong Quan, Xiao-Hong Bai*

2008, 17(2):  163-166. 

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Liquid-phase microextraction with back extraction (LPME-BE) combined with high performance liquid chromatography (HPLC) was investigated for the extraction and determination of magnolol and honokiol in Magnolia officinalis, a traditional Chinese medicine (TCM), and its pharmaceutical preparations, Huo Xiang Zheng Qi peroral liquid and Xiang Sha Yang Wei pellet. Organic solvent, donor and acceptor phases, stirring rate and extraction times were all factors which can influence the efficiency of extraction and were all optimized during the course of this work. Linear calibration curves were obtained in concentration ranges of 1.56-156 μg/mL for magnolol and 1.10-110 μg/mL for honokiol. Detection limits (S/N = 3) were 0.10 and 0.07 μg/mL, respectively. The relative recoveries were both in the range of 98.3% - 105.1% and RSD was lower than 2.5%.

Effect of total flavonoids of epimedium on liver microsomal CYP1A2, CYP3A4 and CYP2E1 activities in rats

Dao-De Hu*, Hui-Juan Yao, Lei Gu, Song-Po Wang, Gao-Lin Liu

2008, 17(2):  167-169. 

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To assess the potential effect of total flavonoids of epimedium (TFE) on cytochrome P450 and activity of its main isoforms in rat liver microsomes. TFE (300 mg/kg) was administered once daily to male Sprague-Dowley rats by gavage for fifteen days. The total cytochrome P450 content and its main isoforms CYP1A2, CYP3A4 and CYP2El activities in rat liver microsomes were detected. The activity of CYP1A2 was measured by fluorometry and the activities of CYP3A4 and CYP2El were determined by measuring the amount of methanal and p-aminophenol formed using UV/Vis spectrophotometer, respectively. Administration of TFE significantly increased the total CYP450 content and activities of CYP1A2, CYP3A4 and CYP2El in rat liver microsomes, compared with the control group. Particularly, the activities of CYP1A2 and CYP2El were enhanced significantly (P<0.01). TFE induced the increase in total CYP450 content and its main isoforms CYP1A2, CYP3A4 and CYP2El activities in rat liver microsomes.