[show abstract][hide abstract] ABSTRACT: This study identified specific and avid RNA aptamers consisting of 2'-hydroxyl or 2'-fluoro pyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of 2'-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. Selected 2'-fluoro aptamer could be truncated up to chemically manufacturable length of 29 nt with increase in the affinity to HCV NS5B. Noticeably, transfection of the minimized aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b sub-genomic and genotype 2a full length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2'-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2'-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.
[show abstract][hide abstract] ABSTRACT: Carcinoembryonic antigen (CEA) is expressed by many types of cancer cells; its overexpression induces cell adhesion, increases resistance to anoikis, and promotes hepatic metastasis of colon cancer cells. The amino acid sequence PELPK in its hinge region, between the N and A1 domains, is required for migration of cancer cells to the liver. We sought to identify ligands of this domain for use in diagnosis and therapy.
We screened for RNA aptamers against the domain of CEA required for metastasis using systematic evolution of ligands by exponential enrichment. The specificity and affinity of the aptamer for CEA protein were characterized by mobility shift, uptake, and surface plasmon resonance assays. We analyzed the effects of the aptamer on metastatic properties of cells, as well as metastasis of colon cancer cells in mice.
Using systematic evolution of ligands by exponential enrichment, we identified an RNA aptamer that bound to the PELPK sequence in CEA with high affinity and specificity. The isolated aptamer bound specifically to CEA-positive cells and inhibited interactions between CEA and heterogeneous nuclear ribonucleoprotein M4. The aptamer inhibited homotypic aggregation, migration, and invasion by CEA-positive cancer cells, but did not affect adhesion of endothelial cells. The aptamer induced colon cancer cell anoikis by interrupting the interaction between death receptor 5 and CEA. The aptamer prevented metastasis of human colon cancer cells to the livers of mice.
An RNA aptamer that binds to the PELPK sequence in CEA inhibits its interactions with heterogeneous nuclear ribonucleoprotein M4 and death receptor 5, migration and invasion by colon cancer cells, and hepatic metastasis of colon cancer cells in mice. It promoted cancer cell anoikis and might be used to identify CEA-positive tumors in patients or be developed as an anti-cancer reagent.
[show abstract][hide abstract] ABSTRACT: Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; 25 µg/ml) and methotrexate (MTX; 50 µg/ml), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptasepolymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.
Korean Journal of Physiology and Pharmacology 02/2012; 16(1):11-6. · 1.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as α3β4, α7 and α9α10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (I(ACh)) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited I(ACh). Pre-treatment of quercetin further inhibited I(ACh) in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of I(ACh) by quercetin was reversible and concentration-dependent. The IC(50) of quercetin was 18.9±1.2 µM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of I(ACh) by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.
Korean Journal of Physiology and Pharmacology 08/2011; 15(4):195-201. · 1.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: The human ether-a-go-go-related gene (HERG) cardiac K(+) channels are one of the representative pharmacological targets for development of drugs against cardiovascular diseases such as arrhythmia. Panax ginseng has been known to exhibit cardioprotective effects. In a previous report we demonstrated that ginsenoside Rg3 regulates HERG K(+) channels by decelerating deactivation. However, little is known about how ginsenoside metabolites regulate HERG K(+) channel activity. In the present study, we examined the effects of ginsenoside metabolites such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) on HERG K(+) channel activity by expressing human α subunits in Xenopus oocytes. CK induced a large persistent deactivating-tail current (Ideactivating-tail ) and significantly decelerated deactivating current decay in a concentration-dependent manner. The EC50 for persistent Ideactivating-tail was 16.6±1.3 μM. In contrast to CK, PPT accelerated deactivating-tail current deactivation. PPD itself had no effects on deactivating-tail currents, whereas PPD inhibited ginsenoside Rg3-induced persistent Ideactivating-tail and accelerated HERG K(+) channel deactivation in a concentration-dependent manner. These results indicate that ginsenoside metabolites exhibit differential regulation on Ideactivating-tail of HERG K(+) channel.
Journal of ginseng research 06/2011; 35(2):191-199. · 2.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ginseng has been used as a general tonic agent to invigorate the human body as an adaptogenic agent. In a previous report, we have shown that ginseng contains a novel glycolipoprotein called gintonin. The main function of gintonin is to transiently enhance intracellular free Ca(2+) [Ca(2+)]i levels in animal cells. The previous method for gintonin isolation included multiple steps using organic solvents. In the present report, we developed a simple method for the preparation of crude gintonin from ginseng root as well as stem and leaf, which produced a higher yield of gintonin than the previous one. The yield of gintonin was 0.20%, 0.29%, and 0.81% from ginseng root, stem, and leaf, respectively. The apparent molecular weight of gintonin isolated from stem and leaf through sodium dodecyl sulfate polyacrylamide gel electrophoresis was almost same as that from root but the compositions of amino acids, carbohydrates or lipids differed slightly between them. We also examined the effects of crude gintonin from ginseng root, stem, and leaf on endogenous Ca(2+)-activated Cl- channel (CaCC) activity of Xenopus oocytes through mobilization of [Ca(2+)]i. We found that the order of potency for the activation of CaCC was ginseng root > stem > leaf. The ED50 was 1.4±1.4, 4.5±5.9, and 3.9±1.1 μg/mL for root, stem and leaf, respectively. In the present study, we demonstrated for the first time that in addition to ginseng root, ginseng stem and leaf also contain gintonin. Gintonin can be prepared from a simple method with higher yield of gintonin from ginseng root, stem, and leaf. Finally, these results demonstrate the possibility that ginseng stem and leaf could also be utilized for ginstonin preparation after a simple procedure, rather than being discarded.
Journal of ginseng research 06/2011; 35(2):209-218. · 2.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: • Remifentanil, an intravenous ultra short-acting opioid, depresses central nervous system activity with an increase in the delta band power, and causes beta activation after discontinuation, resulting in a rebound of the processed electroencephalographic parameters, including 95% spectral edge frequency, the canonical univariate parameter and electroencephalographic approximate entropy. • A sigmoid Emax model, in which the highest predicted values of processed electroencephalographic parameters are restricted to the baseline value, cannot describe a rebound of these parameters. • Electroencephalographic approximate entropy correlated well with the remifentanil blood concentration and demonstrated high baseline stability.
• A combined effect and tolerance model effectively characterized the time course of the remifentanil effect on the central nervous system, including the rebound which occurred during recovery from the remifentanil effect. • Temporal linear mode complexity was comparable with approximate entropy as a univariate electroencephalographic descriptor of the effect of remifentanil on the central nervous system. AIMS Previously, electroencephalographic approximate entropy (ApEn) effectively described both depression of central nervous system (CNS) activity and rebound during and after remifentanil infusion. ApEn is heavily dependent on the record length. Linear mode complexity, which is algorithmatically independent of the record length, was investigated to characterize the effect of remifentanil on the CNS using the combined effect and tolerance, feedback and sigmoid E(max) models. METHODS The remifentanil blood concentrations and electroencephalographic data obtained in our previous study were used. With the recording of the electroencephalogram, remifentanil was infused at a rate of 1, 2, 3, 4, 5, 6, 7 or 8 µg kg(-1) min(-1) for 15-20 min. The areas below (AUC(effect) ) or above (AAC(rebound) ) the effect vs. time curve of temporal linear mode complexity (TLMC) and ApEn were calculated to quantitate the decrease of the CNS activity and rebound. The coefficients of variation (CV) of median baseline (E(0)), maximal (E(max)), and individual median E(0) minus E(max) values of TLMC were compared with those of ApEn. The concentration-TLMC relationship was characterized by population analysis using non-linear mixed effects modelling.
Median AUC(effect) and AAC(rebound) were 1016 and 5.3 (TLMC), 787 and 4.5 (ApEn). The CVs of individual median E(0) minus E(max) were 35.6, 32.5% (TLMC, ApEn). The combined effect and tolerance model demonstrated the lowest Akaike information criteria value and the highest positive predictive value of rebound in tolerance.
The combined effect and tolerance model effectively characterized the time course of TLMC as a surrogate measure of the effect of remifentanil on the CNS.
British Journal of Clinical Pharmacology 01/2011; 71(6):871-85. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate the incidence and severity of injection pain caused by microemulsion propofol and lipid emulsion propofol in relation to plasma bradykinin generation and aqueous free propofol concentrations.
Injection pain was evaluated in 147 patients. Aqueous free propofol concentrations in each formulation, and in formulation mixtures containing agents that reduce propofol-induced pain, were measured by high-performance liquid chromatography. Plasma bradykinin concentrations in both formulations and in their components mixed with blood sampled from six volunteers were measured by radioimmunoassays. Injection pain caused by 8% polyethylene glycol 660 hydroxystearate (PEG660 HS) was evaluated in another 10 volunteers.
The incidence of injection pain [visual analogue scale (VAS) >30 mm] caused by microemulsion and lipid emulsion propofol was 69.7 and 42.3% (P < 0.001), respectively. The median VAS scores for microemulsion and lipid emulsion propofol were 59 and 24 mm, respectively (95% confidence interval for the difference 12.5, 40.0). The aqueous free propofol concentration of microemulsion propofol was seven times higher than that of lipid emulsion propofol. Agents that reduce injection pain did not affect aqueous free propofol concentrations. Microemulsion propofol and 8% PEG660 HS enhanced plasma bradykinin generation, whereas lipid emulsion propofol and lipid solvent did not. PEG660 HS did not cause injection pain.
Higher aqueous free propofol concentrations of microemulsion propofol produce more frequent and severe pain. The plasma kallikrein-kinin system may not be involved, and the agents that reduce injection pain may not act by decreasing aqueous free propofol concentrations.
British Journal of Clinical Pharmacology 12/2008; 67(3):316-25. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: A newly developed microemulsion propofol consisted of 10% purified poloxamer 188 and 0.7% polyethylene glycol 660 hydroxystearate. The authors studied the physicochemical properties, aqueous free propofol concentration, and plasma bradykinin generation. Pharmacokinetics and pharmacodynamics were also evaluated in rats.
The pH, particle size, and osmolarity of microemulsion propofol were measured using a pH meter, particle size analyzer, and cryoscopic osmometer, respectively. The aqueous free propofol and plasma bradykinin were measured by a dialysis method and radioimmunoassay, respectively. Microemulsion propofol was administered by zero-order infusion of 0.5, 1.0, and 1.5 mg . kg . min for 20 min in 30 rats. The electroencephalographic approximate entropy was used as a surrogate measure of propofol effect.
The pH, osmolarity, and particle size of microemulsion propofol are 7.5, 280 mOsm/l, and 67.0 +/- 28.5 nm, respectively. The aqueous free propofol concentration in microemulsion propofol was 63.3 +/- 1.2 mug/ml. When mixed with human blood, microemulsion propofol did not generate bradykinin in plasma. Although microemulsion propofol had nonlinear pharmacokinetics, a two-compartment model with linear pharmacokinetics best described the time course of the propofol concentration as follows: V1 = 0.143 l/kg, k10 = 0.175 min, k12 = 0.126 min, k21 = 0.043 min. The pharmacodynamic parameters in a sigmoid Emax model were as follows: E0 = 1.18, Emax = 0.636, Ce50 = 1.87 mug/ml, gamma = 1.28, ke0 = 1.02 min.
Microemulsion propofol produced a high concentration of free propofol in the aqueous phase. For the applied dose range, microemulsion propofol showed nonlinear pharmacokinetics.
[show abstract][hide abstract] ABSTRACT: The aim of this trial was to evaluate the induction and recovery characteristics of microemulsion propofol (Aquafol; Daewon Pharmaceutical Co., Ltd., Seoul, Korea). Pharmacokinetics, pharmacodynamics, and safety profile were investigated. Lipid emulsion propofol (Diprivan; AstraZeneca, London, United Kingdom) was used as a comparator.
Thirty-one healthy volunteers aged 20-79 yr were given an intravenous bolus of propofol 2 mg/kg, followed by variable rate infusion for 60 min. Each volunteer was studied twice with different formulations at an interval of 1 week. Arterial concentrations of propofol were measured, and Bispectral Index was used as a surrogate measure of propofol effect. The induction and recovery characteristics including bioequivalence were evaluated by noncompartmental analysis. The pharmacokinetics and pharmacodynamics were investigated using a population approach with mixed effects modeling. The rate, severity, and causal relation of adverse events were analyzed.
Both formulations were bioequivalent. The observed time to peak effect after a bolus of both formulations was 1.5 min. Plasma concentration of propofol at loss of consciousness, time to loss of consciousness after a bolus, and time to recovery of consciousness after discontinuation of infusion did not show significant differences. The population pharmacokinetics and pharmacodynamics revealed a variety of differences between two formulations. Aquafol showed similar safety profile to Diprivan.
The efficacy and safety of Aquafol were not different from those of Diprivan within the dose range in this study.