Differential Changes in Functional Activity of Organic Cation Transporters in Rats with Uranyl Nitrate-Induced Acute Renal Failure

맹한주,정석재,심원식,Sun-Joo Ahn,Sang-Soo Yu,Dae-Duk Kim,심창구 대한약학회 2012 Archives of Pharmacal Research Vol.35 No.8

We studied the impact of experimental kidney failure on the pharmacokinetics of a model organic cation and investigated the underlying mechanism(s) of the organic cation transporters. The systemic pharmacokinetics and tissue distribution of triethylmethylammonium (TEMA), a model organic cation, were characterized after intravenous doses of 0.3-30 μmol/kg in rats with or without uranyl nitrate-induced acute renal failure (UN-ARF). To study the effect of endogenous substrates in plasma from UN-ARF rats on organic cation transport, rOCT- or rOCT2-dependent uptake of tetraethylammonium (TEA) was studied in rOCT1-transfected or rOCT2-transfected LLC-PK1 cells, respectively. As a result, the AUC for TEMA was increased, probably because of decreased total clearance, and the tissue-to-plasma concentration ratio (T/P ratio) of TEMA was unchanged in the liver but decreased significantly in the kidneys of UN-ARF rats. In vitro, the uptake of TEA was decreased significantly by adding UN-ARF plasma, compared with control plasma, in rOCT2-overexpressing LLC-PK1 cells, but not in rOCT1-overexpressing LLC-PK1 cells. These observations suggest that the induction of UN-ARF leads to an accumulation of endogenous organic cation(s), probably rOCT2 substrate(s), in the plasma, thereby affecting the TEMA pharmacokinetics and distribution to the kidneys in rats.

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

맹한주,최성업,장동진,이동원,안병락,최민구,송임숙,조관형 한국약제학회 2015 Journal of Pharmaceutical Investigation Vol.45 No.5

A simple, accurate and cost-effective ultraviolethigh 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-fluorenylmethylchloroformate (FMOC), and injected with the UV-HPLC systemfor quantitation. Derivatizedmemantinewere separated on a reverse phase C18 column (Shiseido, 250 × 4.6 mm, 5 lm) with a mixture of 50 mM phosphate buffer (pH 4, adjusted with orthophosphoric acid) and Acetonitrile (20:80, v/v), at a flow rate of 2.0 mL/min.UVdetectionwas monitored at 265 nm. The detector response was specific and linear over the concentration range of 1.0–20.0 lg/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 containingmemantine hydrochloride (i.e., EbixaⓇ, 10 mg). Thus, the developed method would be appropriate for routine in vitro dissolution studies of memantine hydrochloride tablet.

Functional Impairment of P-glycoprotein by Sodium Nitroprusside Pretreatment in Mouse Brain Capillary Endothelial Cells

맹한주,정석재,Yu-Jin Bang 대한약학회 2012 Archives of Pharmacal Research Vol.35 No.7

We examined whether pretreatment of mouse brain blood vessel endothelial cell clone 4 (MBEC4) cells with sodium nitroprusside (SNP), a NOx donor, as an in vitro model of the bloodbrain barrier could affect P-glycoprotein (P-gp) functional activity. Uptake into the cells and MBEC4 plasma membrane vesicles (MPMVs) in the presence or absence of SNP pretreatment was used to investigate functional changes. Increased accumulation of [3H]vincristine, a widely used substrate for P-gp, into MBEC4 was observed upon SNP pretreatment, likely due to impaired P-gp function. To better understand the mechanism of the impairment, MPMVs were prepared and characterized in terms of purity and Na+-dependent glucose uptake. [3H]daunomycin uptake into MPMVs was diminished after SNP pretreatment in the presence of an ATP-regenerating system, indicating that the functional activity of P-gp was impaired after exposure to SNP. Under conditions of excess ATP, daunomycin uptake into the vesicles was still decreased after SNP pretreatment, indicating that SNP interacted directly with the transport system, but not with the ATP-regenerating system. Together, these results suggest that NO or NOx functionally impairs P-gp in the in vitro blood-brain barrier model with SNP pretreatment.

Toxicological Relevance of Transporters

맹한주,정석재 한국독성학회 2007 Toxicological Research Vol.23 No.1

Transporters are membrane proteins that mediate the transfer of substrate across the cellular membrane. In this overview, the characteristics and the toxicological relevance were discussed for various types of transporters. For drug transporters, the overview focused on ATP-binding cassette transporters and solute carrier family 21A/22A member transporters. Except for OCTN transporters and OATP transporters, drug transporters tend to have broad substrate specificity, suggesting drugdrug interaction at the level of transport processes (e.g., interaction between methotrexate and nonsteroidal anti-inflammatory agents) is likely. For metal transporters, transporters for zinc, copper and multiple metals were discussed in this overview. These metal transporters have comparatively narrow substrate specificity, except for multiple metal transporters, suggesting that inter-substrate interaction at the level of transport is less likely. In contrast, the expressions of the transporters are often regulated by their substrates, suggesting cellular adaptation mechanism exists for these transporters. The drug-drug interactions in drug transporters and the cellular adaptation mechanisms for metal transporters are likely to lead to alterations in pharmacokinetics and cellular metal homeostasis, which may be linked to the development of toxicity. Therefore, the transporter-mediated alterations may have toxicological relevance.

Liquid chromatography–tandem mass spectrometry for quantification of cefaclor in rat plasma and its application to pharmacokinetic studies

진효언,맹한주,김유철 한국약제학회 2014 Journal of Pharmaceutical Investigation Vol.44 No.2

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the positiveelectrospray ionization mode was developed and validatedin order to analyze cefaclor, a second generation cephalosporinantibiotic, in rat plasma. The plasma was pre-treatedby a single step of protein precipitation with methanol, andthen injected directly into the LC/MS/MS system forquantification. Drugs were separated on a Synergi 4lPolar-RP 80 A column (150 9 2.0 mm) with a mixture of0.1 % formic acid and methanol (65 : 35, v/v) as the mobilephase at 0.2 mL/min. Detection was performed with multiplereaction-monitoring modes at m/z 368.1 ? 174.2 (forcefaclor) and m/z 396.0 ? 227.1 (for cefdinir, the internalstandard). The limit of quantification (LOQ) was determinedto be 10 ng/mL with acceptable linearity rangingfrom 10 to 10,000 ng/mL. Validation parameters for cefaclor,including accuracy, precision, absolute matrix effect,and stability in rat plasma, were acceptable according tothe assay validation guidelines of the FDA (U.S. Departmentof Health & Human Services, Food and DrugAdministration, Guidance for Industry, BioanalyticalMethod Validation, 2001). The developed analyticalmethod was successfully applied to the pharmacokineticstudies of cefaclor in rats. These observations suggest,therefore, that the validated assay can be used in routinepharmacokinetic studies of cefaclor in rats.

Contribution of CNT1 and ENT1 to ribavirin uptake in human hepatocytes

최민구,김민혜,맹한주,송임숙 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.5

The objective of this study was to investigate thecontributions of a sodium-dependent concentrative nucleosidetransporter (CNT) 1 and an equilibrative nucleosidetransporter (ENT) 1 to ribavirin uptake in human hepatocytes. The initial studies in oocytes expressing CNT1 andENT1 showed increases in ribavirin uptake, indicating thatribavirin was a substrate for both CNT1 and ENT1. TheCNT1- and ENT1-mediated ribavirin uptake showed concentrationdependency with the following kinetics parameters:Km 26.3 lM and Vmax 426.2 fmol/min/oocyte forCNT1; Km 70.5 lM and Vmax 134.3 fmol/min/oocyte forENT1. Ribavirin uptake clearance in six human hepatocytesranged from 21.3 to 300.7 lL/min. Estimation of the contributionsof CNT1 and ENT1 to the hepatic uptake ofribavirin by using a relative activity factor method indicatedthat the relative contribution of ENT1 to the ribavirin uptakewas 82.8 ± 3.9 %. Real-time polymerase chain reactionanalysis of CNT1 and ENT1 expressions in the hepatocytesshowed that ENT1 mRNA expression was closely correlatedwith ribavirin uptake (R = 0.95, P = 0.003) while CNT1was not. The findings indicated that ENT1 was the majortransporter controlling the hepatic uptake of ribavirin.

Therapeutic targets of vitamin D receptor ligands and their pharmacokinetic effects by modulation of transporters and metabolic enzymes

최민식,김유철,맹한주 한국약제학회 2020 Journal of Pharmaceutical Investigation Vol.50 No.1

Vitamin D is involved in retaining the balance of several minerals in the body, such as calcium and phosphate, which are crucial for the bone formation and development. These physiological effects of vitamin D are mediated by activation of the transcription factor vitamin D receptor (VDR). Moreover, vitamin D is closely related to several pathological conditions including osteoporosis, secondary hyperparathyroidism, cancer, psoriasis and autoimmune diseases, which is not only due to calcemic but also non-calcemic effects of vitamin D such as cell proliferation, differentiation, and immunomodulation. These various abilities of vitamin D have made VDR an attractive therapeutic target. Already, numerous vitamin D analogs have been developed and studied in in vitro disease models or animal models, and some have been in clinical trials or approved for the treatment of certain diseases. In addition, the transcriptional and/or post-transcriptional regulation by VDR activation also affects the genes involved in drug metabolism and disposition, possibly leading to pharmacokinetic changes of several drugs in clinical use. This review provides a detailed summary of therapeutic targets of VDR ligands, and their effects on the transporters and metabolic enzymes, causing in vitro and in vivo pharmacokinetic changes of several drugs. The clinical research is further required for the confirming the clinical relevance of pharmacokinetic drug interactions by VDR ligands.

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

Ajay Vijayakumar,Rengarajan Baskaran,맹한주,유봉규 대한약학회 2017 Archives of Pharmacal Research Vol.40 No.7

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 300–500 nm and stayed submicron size up to 12 months. 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 1 h. When the G-NLC dispersion was orally administered to rats, Cmax of the free curcumin was 15.2 and 32.3 ng/mL at doses of 50 and 100 mg/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 shelflife 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.

Designing Tyrosinase siRNAs by Multiple Prediction Algorithms and Evaluation of Their Anti-Melanogenic Effects

권옥선,권수정,김진상,이건봉,맹한주,이정미,황귀서,차혁진,천광훈 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.3

Melanin is a pigment produced from tyrosine in melanocytes. Although melanin has a protective role against UVB radiationinduced damage, it is also associated with the development of melanoma and darker skin tone. Tyrosinase is a key enzyme in melanin synthesis, which regulates the rate-limiting step during conversion of tyrosine into DOPA and dopaquinone. To develop effective RNA interference therapeutics, we designed a melanin siRNA pool by applying multiple prediction programs to reduce human tyrosinase levels. First, 272 siRNAs passed the target accessibility evaluation using the RNAxs program. Then we selected 34 siRNA sequences with ΔG ≥-34.6 kcal/mol, i-Score value ≥65, and siRNA scales score ≤30. siRNAs were designed as 19-bp RNA duplexes with an asymmetric 3’ overhang at the 3’ end of the antisense strand. We tested if these siRNAs effectively reduced tyrosinase gene expression using qRT-PCR and found that 17 siRNA sequences were more effective than commercially available siRNA. Three siRNAs further tested showed an effective visual color change in MNT-1 human cells without cytotoxic effects, indicating these sequences are anti-melanogenic. Our study revealed that human tyrosinase siRNAs could be efficiently designed using multiple prediction algorithms.

Isolation and Identification of Steroidogenic Peptides from Calf Spleen

지준필,김종국,진수언,반은미,이효종,박유미,박요한,맹한주,김형태 대한약학회 2012 Archives of Pharmacal Research Vol.35 No.4

Since women with climacteric syndrome have significantly lower serum levels of estradiol and other related hormones, hormone replacement therapies (HRT) such as estrogen are needed to lessen symptoms. However, HRT can often cause severe adverse effects that include many cancers and stroke. Therefore, new and novel approaches to relieve climacteric syndrome still need to be developed. The aim of this study was to identify biologically active peptides from calf spleen that are responsible for stimulating biosynthesis of steroid hormone and to explore the potential of isolated peptides as therapeutic agents for menopausal syndrome. The reverse phase HPLC system was used to isolate active compounds from calf spleen extract, a cell culture system was used to screen the activity of stimulating hormone secretion, and Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry was used for molecular weight determination. In the present study, two calf steroidogenic peptides, CSP-1 (MW; 4.655 kDa) and CSP-2 (MW; 8.331 kDa), were isolated and identified from calf spleen and may be putative climacteric syndrome therapeutic agents.