Han-Joo Maeng

Quercetin Directly Interacts with Vitamin D Receptor (VDR): Structural Implication of VDR Activation by Quercetin

The vitamin D receptor (VDR) is a member of the nuclear receptor (NR) superfamily. The VDR binds to active vitamin D3 metabolites, which stimulates downstream transduction signaling involved in various physiological activities such as calcium homeostasis, bone mineralization, and cell differentiation. Quercetin is a widely distributed flavonoid in nature that is known to enhance transactivation of VDR target genes. However, the detailed molecular mechanism underlying VDR activation by quercetin is not well understood. We first demonstrated the interaction between quercetin and the VDR at the molecular level by using fluorescence quenching and saturation transfer difference (STD) NMR experiments. The dissociation constant (Kd) of quercetin and the VDR was 21.15 ± 4.31 μM, and the mapping of quercetin subsites for VDR binding was performed using STD-NMR. The binding mode of quercetin was investigated by a docking study combined with molecular dynamics (MD) simulation. Quercetin might serve as a scaffold for the development of VDR modulators with selective biological activities.

Development and validation of a highly sensitive LC–MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study

A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC–MS/MS). A minimal volume of human plasma sample (20 μL) was prepared by simple deproteinization with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C18 column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1xa0% formic acid at a flow rate of 0.3xa0mL/min over a total run time of 2.0xa0min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22xa0→xa0242.17 for acacetin and m/z 277.59xa0→xa0175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1–500xa0ng/mL with a lower limit of quantitation of 0.1xa0ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9xa0%, and the intra- and inter-day accuracy ranged from 96.8 to 108xa0%. Mean recovery of acacetin in human plasma was within the range of 91.5–95.6xa0%. This validated LC–MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.

Specific Inhibition of the Distribution of Lobeglitazone to the Liver by Atorvastatin in Rats: Evidence for a Rat Organic Anion Transporting Polypeptide 1B2–Mediated Interaction in Hepatic Transport

Cytochrome P450 enzymes and human organic anion transporting polypeptide (OATP) 1B1 are reported to be involved in the pharmacokinetics of lobeglitazone (LB), a new peroxisome proliferator–activated receptor γ agonist. Atorvastatin (ATV), a substrate for CYP3A and human OATP1B1, is likely to be coadministered with LB in patients with the metabolic syndrome. We report herein on a study of potential interactions between LB and ATV in rats. When LB was administered intravenously with ATV, the systemic clearance and volume of distribution at steady state for LB remained unchanged (2.67 ± 0.63 ml/min per kg and 289 ± 20 ml/kg, respectively), compared with that of LB without ATV (2.34 ± 0.37 ml/min per kg and 271 ± 20 ml/kg, respectively). Although the tissue-to-plasma partition coefficient (Kp) of LB was not affected by ATV in most major tissues, the liver Kp for LB was decreased by ATV coadministration. Steady-state liver Kp values for three levels of LB were significantly decreased as a result of ATV coadministration. LB uptake was inhibited by ATV in rat OATP1B2-overexpressing Madin–Darby canine kidney cells and in isolated rat hepatocytes in vitro. After incorporating the kinetic parameters for the in vitro studies into a physiologically based pharmacokinetics model, the characteristics of LB distribution to the liver were consistent with the findings of the in vivo study. It thus appears that the distribution of LB to the liver is mediated by the hepatic uptake of transporters such as rat OATP1B2, and carrier-mediated transport is involved in the liver-specific drug–drug interaction between LB and ATV in vivo.

Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug

In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (1H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter–positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

The identification of lobeglitazone metabolites in rat liver microsomes and the kinetics of the in vivo formation of the major metabolite M1 in rats.

The objective of this study was to elucidate the chemical structure of the metabolites derived from lobeglitazone (LB) during its incubation with rat liver microsomes and to characterize the kinetics of formation of the major metabolite M1 in vivo. Using high performance liquid chromatography coupled with a hybrid quadrupole linear ion trap, the metabolites were derived from LB during its incubation with rat liver microsomes. From various fragmentation patterns obtained from the metabolites, LB was biotransformed into 5 metabolites in the incubation, in which demethylation and hydroxylation appeared to be the principle metabolic pathways in vitro; Amongst the five primary metabolites, M1, a demethylated derivative of LB, appeared to be the major metabolite of LB, based on a comparison on the peak intensities in the ion chromatogram. In a study of the in vivo kinetics of formation of M1 in rats, the rate of formation of M1 from LB was determined to be 0.252 and 0.216mL/min/kg at doses of 0.5mg/kg and 2mg/kg of LB, respectively, suggesting that the kinetics of M1 formation were linear in the dose range tested. Considering the fact that LB is primarily eliminated by hepatic metabolism in rats, the formation of M1 accounts for approximately 7.50-9.76% of the overall elimination of LB in rats.

Quantification of mequitazine in human plasma by gas chromatography– quadrupole mass spectrometry and its application to a human pharmacokinetic study

A specific and sensitive gas chromatography-mass spectrometry (GC-MS) with quadrupole mass analyzer type was developed and validated for the quantitative analysis of mequitazine in human plasma. After liquid-liquid extraction of plasma samples containing mequitazine and promethazine (internal standard, IS) using hexane with pH adjustment, the extract was evaporated and an aliquot of reconstituted residue was injected into the GC-MS system. The assay showed linearity over a concentration range from 1 to 50u2009ng/mL. Intra- and inter-day precision for mequitazine was <9.09 and 9.29%, respectively, and intra- and inter-day accuracy ranged from -7.97 to 9.05% and from -1.51 to 7.89%, respectively. The lower limit of quantification was 1u2009ng/mL in the present assay. The developed analytical method was successfully applied to a pharmacokinetic study after a single oral administration of mequitazine in human subjects.

Vitamin D Receptor-Mediated Upregulation of CYP3A4 and MDR1 by Quercetin in Caco-2 cells

To examine whether quercetin interacts with vitamin D receptor, we investigated the effects of quercetin on vitamin D receptor activity in human intestinal Caco-2 cells. The effects of quercetin on the expression of the vitamin D receptor target genes, vitamin D3 24-hydroxylase, cytochrome P450 3A4, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were measured using quantitative polymerase chain reaction. The vitamin D receptor siRNA was used to assess the involvement of the vitamin D receptor. Vitamin D receptor activation using a vitamin D responsive element-mediated cytochrome P450 3A4 reporter gene assay was investigated in Caco-2 cells transfected with human vitamin D receptor. We also studied the magnitude of the vitamin D receptor activation and/or synergism between 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and quercetin-like flavonoids. Slight but significant increases in the mRNA expression of cytochrome P450 3A4, vitamin D3 24-hydroxylase, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were observed after 3 days of continual quercetin treatment. The silencing effect of vitamin D receptor by vitamin D receptor siRNA in Caco-2 cells significantly attenuated the induction of the vitamin D receptor target genes. Moreover, quercetin significantly enhanced cytochrome P450 3A4 reporter activity in Caco-2 cells in a dose-dependent manner, and the expression of exogenous vitamin D receptor further stimulated the vitamin D receptor activity. Quercetin-like flavonoids such as kaempferol stimulated the vitamin D receptor activity in a manner similar to that seen with quercetin. Taken together, the data indicates that quercetin upregulates cytochrome P450 3A4 and multidrug resistance protein 1 expression in Caco-2 cells likely via a vitamin D receptor-dependent pathway.

Validation and application of a simple reverse phase HPLC method for in vitro dissolution studies of memantine hydrochloride tablet

A simple, accurate and cost-effective ultraviolet-high performance liquid chromatography (UV-HPLC) assay method was developed and validated for the determination of derivatized memantine, a representative oral noncompetitive N-methyl-d-aspartate receptor antagonist to treat Alzheimer’s disease, in dissolution medium. Optimized derivatization process of memantine was performed with 9-fluorenylmethyl-chloroformate (FMOC), and injected with the UV-HPLC system for quantitation. Derivatized memantine were separated on a reverse phase C18 column (Shiseido, 250xa0×xa04.6xa0mm, 5xa0μm) with a mixture of 50xa0mM phosphate buffer (pH 4, adjusted with orthophosphoric acid) and Acetonitrile (20:80, v/v), at a flow rate of 2.0xa0mL/min. UV detection was monitored at 265xa0nm. The detector response was specific and linear over the concentration range of 1.0–20.0xa0μg/mL. Validation parameters of derivatized memantine with the sensitivity, selectivity, linearity, accuracy, precision and stability in dissolution medium (pH 1.2) were acceptable based on International Conference on Harmonization Q2 (R1). The assay method validated in this work was successfully applied for a dissolution study of a commercial tablet containing memantine hydrochloride (i.e., Ebixa®, 10xa0mg). Thus, the developed method would be appropriate for routine in vitro dissolution studies of memantine hydrochloride tablet.

Feasibility of the functional expression of the human organic anion transporting polypeptide 1B1 (OATP1B1) and its genetic variant 521T/C in the mouse liver

&NA; The objective of this study was to examine the feasibility of functional expression of the human organic anion transporting polypeptide 1B1 (hOATP1B1) forms in the liver of the mouse. After the mouse received the gene of interest (i.e., luciferase as the reporter or hOATP1B1) via hydrodynamic gene delivery (HGD) method, the expression was found to be liver‐specific while alterations in the serum biochemistry and hepatocyte histology were apparently transient and reversible. The reporter activity was also detected in the plasma, but not in the blood cell in mice that received HGD, suggesting that the protein is probably released due to transiently increased permeability in hepatocytes by HGD. Using this delivery condition, the expression of hOATP1B1 was readily detected in the liver, but not in other tissues, of the mice receiving HGD for the transporter gene. Compared with the sham control mice, the uptake of pravastatin into the liver increased significantly in mice receiving hOATP1B1 wild type; the uptake parameters decreased consistently in mice expressing the 521T>C variant compared with that of the wild type control. These observations suggest that the functional expression of human transporter gene in mice is feasible, further suggesting that this treatment is practically useful in the pharmacokinetic studies for hOATP1B1 substrates. Graphical abstract Figure. No caption available.

Ginsenoside improves physicochemical properties and bioavailability of curcumin-loaded nanostructured lipid carrier

The aim of this study was to develop a ginsenoside-modified nanostructured lipid carrier (G-NLC) dispersion containing curcumin. The NLC was prepared by melt emulsification with slight modification process. Different G-NLC dispersion systems were prepared using lipid carrier matrix composed of ginsenoside, phosphatidylcholine, lysophosphatidylcholine, and hydrogenated bean oil. TEM image of the nanoparticles in the NLC dispersion showed core/shell structure, and there was corona-like layer surrounding the particles in the G-NLC. The mean particle size of G-NLC dispersion was in the range of about 300–500xa0nm and stayed submicron size up to 12xa0months. The in vitro release of curcumin was faster in pH 1.2 compared to pH 6.8 and it showed linear release pattern after lag time of 1xa0h. When the G-NLC dispersion was orally administered to rats, Cmax of the free curcumin was 15.2 and 32.3xa0ng/mL at doses of 50 and 100xa0mg/kg, respectively, while it was below quantification limit when curcumin was administered as of dispersion in distilled water. Based on these results, it is certain that ginsenoside modulated the NLC dispersion, leading to enduring shelf-life of the dispersion system and enhanced bioavailability. These results strongly suggest that ginsenoside holds a promising potential as a pharmaceutical excipient in the pharmaceutical industries to increase the utility of various bioactives.