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The 1st Japan-Korea Joint Workshop@on Graduation Research Presentation in Okayama 2013
Program



Poster presentations at OU Booth (Lecture room 1)
Curator: Assoc. Prof. Manabu Suno


The 1st group (9:30-11:30)

OU-A1   Tatsuya Sakiyama (Pharmaceutical Formulation Design)
Synchronous-Dual-Zero-Net-Flux Microdialysis for Monitoring Focal Unbound Drug Concentration Profiles: Improvement of Monitoring Lag.

OU-A2   Mai Takahashi (Pharmaceutical Formulation Design)
Oral amlodipine for dogs: Pharmaceutical modification to suppress its bitterness

OU-A3   Miki Terasaki (The Center for Development of Advanced Pharmaceutical Education)
Effect of stem lettuce on allergic rhinitis model in mice

OU-A4   Kenta Yagi (Clinical Pharmacy)
Differential synergistic effect of COX inhibitors on cell survival suppressed by Sorafenib in hepatocellular cancer cells

OU-A5   Yagi Shimpei (Clinical Pharmacy)
Influence of inflammation on diazepam-induced sleep latency and anti-anxiety effect in mice

OU-A6   Yoneda SaoriiClinical Pharmacyj      
Effect of romiplostim on impaired spatial memory in a rat model of chemotherapy

OU-A7   Hiroko Nakamura (Clinical Pharmacy)      
Ketamine exerts antidepressant - like effects in ACTH-treated rats

OU-A8   Maki Okino (Clinical Pharmacy)      
The antitumor effect of COX inhibitors combined with Doxorubicin

OU-A9   Atsuyoshi Okada (Clinical Pharmacokinetics and Therapeutics)      
Evaluation of the CNS drug sensitivity in rats with acute renal failure

OU-A10   Shunsuke Kanetada (Biopharmaceutics)
Determinants for in-vivo anti-tumor effects of liposomal doxorubicin (DOX)

OU-A11   Yoshihiro Sakuwa (biopharmaceutics)      
Contribution of P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) to Intestinal First-Pass Effect of Loperamide in Rats

OU-A12   Takamori Aiko (Biopharmaceutics)      
Basic Study for Development of New Absorption Enhancing Formulation using Polyamine Derivatives

OU-A13   Naoki Kishi (Health Chemistry)
Function of primate UDP-glucuronosyltransferases expressed in gastrointestinal tract

OU-A14   Yu Kinashi (Health Chemistry)
Human UDP-glucuronosyltransferase isoforms involved in the glucuronidation of mono (2-ethylhexyl) phthalate

OU-A15   Marina Mukai (Health Chemistry)      
Species and Gender Differences in Propofol Glucuronidation

OU-A16   Satoko Watanabe (Synthetic and Medicinal Chemistry)      
Synthesis of Cytotoxic Tannin Analogs for Tumor Cells

OU-A17   Aya Takeuchi (Synthetic and Medicinal Chemistry)      
Synthesis and Anti-MRSA Activity of Indoloquinoline Derivatives

OU-A18   Mitsugu Bando (Creative Medicinal Chemistry)      
Synthesis of Optically Active Phenylpropanoic Derivatives with Fluorinated Quaternary Carbon Atom as PPAR? Agonists

OU-A19   Yuta Tanaka (Creative Medicinal Chemistry)      
Design and Synthesis of Novel PPAR? Agonists based on MD Simulations

OU-A20   Satoko AkitaiNatural Product Chemistryj      
Effects of Tannins and Alkyl Gallates on Aeromonas sobria

OU-A21   Yui Arima (Natural Product Chemistry)      
Constituents of White Adzuki Bean

OU-A22   Yasuyo Okame (Natural Product Chemistry)      
Suppressing Effect of Tannins from Strawberry Leaves on the Norfloxacin Resistance of MRSA


The 2nd group (13:00-15:00)

OU-B1   Watanabe Shinji (Protein Function)      
Mutagenesis induced by chloroethylating anticancer drug, and its restoration mechanism

OU-B2   Hiroki Watanabe (International Joint Research Center for Drug Discovery on Intractable infectious Diseases)
Analysis of switching mechanisms in two cell-death types, necrosis and apoptosis

OU-B3   Tetsuya Yamashita (Protein Function)      
Inducible effect of skim milk on the production of serine protease of Aeromonas trota

OU-B4   Akinori Maruo (Protein Function)      
Studies on the purification of the intermediate of Aeromonas metalloprotease

OU-B5   Noriko Tanaka (Protein Function)      
Genotoxicity of UVA activated N-nitrosoproline on human derived keratinocytes

OU-B6   Ayako Maekawa (Biophysical Chemistry)      
Permeability Enhancement of the Outer Membrane of Escherichia coli as Investigated by the Rose Bengal-Induced Photoinactivation of Bacteria

OU-B7   Yuko Manabe (Biophysical Chemistry)     
A study on hippocampal long-term potentiation in awake mice

OU-B8   Yuka Nakanishi (Biophysical Chemistry)    
Porphyrin-Induced Photoinactication of Bacteria and Membrane Damage of Erythrocytes

OU-B9   Miho Hyodo (Biophysical Chemistry)    
Action of Amphipathic Peptides on Bacterial Cell Membranes
|Relationship between Antibacterial Activity and Membrane Permeability|

OU-B10   Aya Kakinoki (Immulobiology)   
Establishment and characterization of a culture model of murine mucosal mast cells

OU-B11   Satoshi Hokari (Immunobiology)    
Expression of neurotrophin receptors in murine mast cells

OU-B12   Yohei Manabe (Immunobiology)           
Halogenated dinitrobenzenes induce degranulation of rat peritoneal mast cells


OU-B13   Hiroko Morinaga (Sanitary microbiology)
The genes of Cupriavidus metallidurans PD11 strain which are expressed in degradation of dichloromethane

OU-B14   Hayata Dodan (Sanitary microbiology)     
 
Investigation of expression control mechanism of RND-type multidrug efflux pump in Klebsiella pneumoniae

OU-B15   Hanako Sunouchi (Sanitary microbiology)     
The mobility of the genomic region around the metalloprotease gene (vvp) in Vibrio vulnificus

OU-B16   Megumi Kurata (Sanitary microbiology)
Typing of Vibrio vulnificus by genes encoding the hemolysin and protease

OU-B17   Tsuyoshi Ito (Emergency Pharmaceutics)      
Investigation of Tubal Ingestion Method for Kampo Medicine in Advanced Critical Care Center

OU-B18   Yuka Kayano (Emergency Pharmaceutics)      
Risk Factor of Acute Renal Failure Induced by Edaravone in Patients with Cerebrovascular Disorder

OU-B19   Asuka Shiomi (Emergency Pharmaceutics)     
Risk Factor of Hypotension Induced by Dexmedetomidine in the Advanced Critical Care and Emergency Center

OU-B20   Masahiko Asano (Oncology Pharmaceutical Care)     
Determination of Platinating Agents in Human Plasma

OU-B21   Takahiro Ishino (Oncology Pharmaceutical Care)     
Photodegradation of (+)-tramadol and Stability of Oral Liquid Preparations of Tramadol

OU-B22   Kenta Sakamoto (Oncology Pharmaceutical Care)      
Determination of sirolimus in human plasma




Poster presentations at SKKU Booth (Lecture room 1)

Curator: Assoc. Prof. Yukio Sugimoto

The 1st group (9:30-11:30)

SKKU-1   Min Sang Lee (Molecular Pharmaceutics)      
Enhanced Transfection by Antioxidative Polymeric Gene Carrier that Reduces Polyplex-Mediated Cellular Oxidative Stress

SKKU-2   Jihye Park      
Pd(II)-Catalyzed Decarboxylative Acylation of Phenylacetamides with ƒ¿-oxocarboxylic acids and Application to the Synthesis of 3-Isochromanone

SKKU-3   Da-Young Shin (Environmental toxicology and Preventive pharmacy)      
Polyhexamethyleneguanidine phosphate induced oxidative stress-mediated DNA damage in human A549 lung adenocarcinoma cell line

SKKU-4   Kyu-Mok Hwang (Physical Pharmacy)     
Preparation of galantamine drug-in-adhesive patch and investigation of formulation factors affecting its skin permeation rate

The 2nd group (13:00-15:00)

SKKU-5   Youngju Kim (Medicinal Chemistry)  
Identification of Ligands for HIV-1 RNA Stem-loop as -1 Ribosomal Frameshift Modulators by In Silico Screening

SKKU-6   Seung Han Seon     
Macrophages play a key role in the protection by Streptococcus pneumoniae pep27 mutant vaccine

SKKU-7   Sang-Joon Kim (Physical Pharmacy)     
Investigation of Formulation Factors for Doxazosin Mesylate Pellets

* All posters in the SKKU booth will be placed full-time.



SKKU-1 

Enhanced Transfection by Antioxidative Polymeric Gene Carrier that Reduces Polyplex-Mediated Cellular Oxidative Stress
Min Sang Lee (Molecular Pharmaceutics Lab)

Object  The object of this study was to enhance transfection efficiency using an antioxidative transfection system minimizing cellular oxidative stress
Methods  PEI-PLGA was synthesized and used as a gene carrier containing hydrophobic antioxidant. Cellular oxidative stress and mitochondrial membrane potential (ƒ¢ƒµ) were measured by using a H2DCFDA and JC-1, respectively. Transfection efficiency was determined by measuring a reporter gene expression level.
Results and Discussion  The PEI-PLGA carried out the simultaneous delivery of antioxidant and plasmid DNA, resulting in a reduction in cellular ROS generation and helped to maintain ƒ¢ƒµ. In addition, the transfection efficiency was dramatically increased.
Conclusion  An antioxidative polymeric gene carrier has great potential as a novel nonviral vector for gene delivery.




SKKU-2 

Pd(II)-Catalyzed Decarboxylative Acylation of Phenylacetamides with ƒ¿-oxocarboxylic acids and Application to the Synthesis of 3-Isochromanone
Jihye Park and In Su Kim

The development of new carbon-carbon bond-forming reactions continues to be an essential goal in organic chemistry. Traditional metal-catalyzed cross-coupling reactions between aryl metal reagents and aryl halides are well-established methods for the construction of C?C bonds and synthesis of complex molecules. Recently, transition-metal-catalyzed decarboxylative cross-coupling reactions using aryl carboxylic acids as aryl surrogates have received much attention since such transformations provide new opportunities to use readily available carboxylic acids as starting materials for organic synthesis.
Transition-metal-catalyzed oxidative acylation of sp2 C?H bonds in aromatic compounds with various directing groups, e.g., pyridines, oximes, acetanilides, and indole, with aldehydes or alcohols were reported. However, decarboxylative C?H bond acylations using ƒ¿-oxocarboxylic acids as acyl surrogates were relatively unexplored. Goossen first demonstrated a palladium-catalyzed decarboxylative crosscoupling reaction of aryl bromides with ƒ¿-keto carboxylate salts as acyl anion equivalents to afford diaryl ketones. Ge described elegant studies on a palladium-catalyzed decarboxylative acylation of acetanilides and phenylpyridines with ƒ¿-oxocarboxylic acids as acyl sources via C?H bond activation. Recently, Guo and Duan described a decarboxylative acylation of cyclic enamides with ƒ¿-oxocarboxylic acids to provide ƒÀ-acyl enamides.
Herein we described our recent result on a Pd-catalyzed decarboxylative ortho-acylation of O-methyl ketoximes and phenylacetamides with ƒ¿-keto acids via C?H bond activation. This protocol provides an efficient access to a range of ortho-acyl phenylacetamides, which can be easily converted to 3-isochromanone derivatives.



SKKU-3 

Polyhexamethyleneguanidine phosphate induced oxidative stress-mediated DNA damage in human A549 lung adenocarcinoma cell line
Da-Young Shin (Laboratory of Environmental toxicology and Preventive pharmacy)

[Object] Recently household chemicals exposure by inhalation has been increased and its health effects became emerging issue. Especially, in Korea, the major component of humidifier disinfectant, polyhexamethy- leneguanidine phosphate (PHMG) caused deaths through lung fibrosis. However, the mechanism under the fibrosis is still unknown. In this study, the oxidative stress and DNA damage of PHMG was investigated in human A549 lung adenocarcinoma cell line, to study the potential role of DNA damage in PHMG-induced fibrosis.
[Methods] First, the cytotoxic effect of PHMG was assessed by WST-1 assay. Second, cells were exposed to PHMG for 6 hours after which ROS generation was measured by DCF-DA assay. Lastly, Oxidative stress-mediated DNA damage by PHMG was studied via modified comet assay.
[Results and Discussion] After 24 h exposure, cell viability was dose-dependently reduced by PHMG, and partially increased with the treatment of deferoxamine. PHMG significantly promoted ROS generation and this is partially inhibited by N-acetyl-L-cysteine. In addition, oxidative DNA damage was detected in cells exposed to PHMG.
[Conclusion] In conclusion, the PHMG induced oxidative stress in human lung cells, and this effect can be followed by DNA damage. Through this study, the potential role of DNA damage in PHMG-induced fibrosis was observed. Further, expression of specific genes related to fibrosis will be assessed.



SKKU-4

Preparation of galantamine drug-in-adhesive patch and investigation of formulation factors affecting its skin permeation rate
Kyu-Mok Hwang (Physical Pharmacy Lab.)

[Object] To prepare galantamine drug-in-adhesive (GLT DIA) transdermal patch and to investigate the effect of API salt form, concentration and type of pressure sensitive adhesives (PSA) on its skin permeation rate.
[Methods] The target permeation rate of galanatmine through the skin was decided by using a simulation software (ScientistR, MicroMath Research, USA). The GLT DIA patches were prepared by dissolving the drug into ethanol and then mixing with PSA solution using mechanical stirrer. The drug-PSA solution was coated on a polyester released liner using a coating unit (Labcoater LTE-S, Mathis AGE, Oberhasli, Switzerland) and laminated with a polyester backing film. In vitro skin permeation test was done using Franz diffusion cells. In vivo transdermal drug delivery was done after application to New Zealand white rabbits weighing 1.8-2 kg. The amounts of galantamine in plasma or receptor medium were determined by a validated HPLC method. The correlation of in vivo permeation rate and in vitro permeation rate by type of PSA was also determined.
[Results and Discussion] The target permeation rate of GLT DIA was between 10 ƒÊg/cm2Eh and 100 ƒÊg/cm2Eh considering the interspecies differences between human and animals. Acrylate PSA was divided into three groups according to their functional groups: carboxyl group, hydroxyl group, and no functional group. Among them, the PSA group having hydroxyl functional group, and especially DT-2510 showed the highest permeation rate (12.60 } 3.31 ƒÊg/cm2Eh). This result had similar result in rank with in vivo study on rabbits, in which AUC0-24 by PSA types is written in decreasing order: hydroxyl group, no functional group, carboxyl group. This can be explained by less drug-polymer interaction, resulting in faster drug diffusion rates. The concentration of drug in GLT DIA patches was proportional to the permeation rate, which is explained by the Fickfs diffusion law. The prepared GLT DIA showed better pharmacokinetic properties compared to oral solution or IV injection. The mean residence time of all GLT DIA patches ( -OH: 8.40 } 2.87 h, -COOH: 17.64 } 3.01 h, None: 15.07 } 2.08 h) were higher than IV injection (0.37}0.05 h) and oral solution 0.85 }0.18 h). The absolute bioavailability GLT DIA( -OH: 80.30%, -COOH: 41.78%, None: 55.98%)was also higher than oral solution (21.52%)
[Conclusion] GLT DIA patches have advantages over oral solution in terms of bioavailability and MRT which results in higher patient compliance. The high correlation of in vivo profiles and in vitro profile by type of PSA can be a useful tool in determining selection of PSA at preliminary investigation.




SKKU-5 

Macrophages play a key role in the protection by Streptococcus pneumoniae pep27 mutant vaccine
Youngju Kim,a* Junghyun Chae,b So-Jung Park,a and Hyun-Ju Parka

aMedicinal Chemistry Laboraotry, School of Pharmacy, Sungkyunkwan University,
bDepartment of Chemistry, Sungshin Womenfs University

[Object] The aim of this project is to discover novel ligands for HIV-1 RNA stem-loop by structure-based virtual screening of chemical database. HIV-1 requires a programmed -1 ribosomal frameshifting (-1 RF) for expressing Gag and Gag-Pol fusion protein, of which ratio is critical for HIV-1 replication. The -1 RF is induced by HIV-1 RNA stem-loop and the stability of stem-loop is important in maintaining efficient -1 RF. Thus, a small molecule that changes the -1 RF efficiency by interacting with RNA stem-loop, can be developed as an anti- HIV-1 agent.
[Methods] To select the candidate ligands targeting HIV-1 RNA stem-loop, first, the Unity 3D (Sybyl-X 2.0, Tripos Inc.) search of chemical database including ZINC and in-house was carried out using a pharmacophore query. This query was established based on the NMR solution structure of HIV-1 RNA stem-loop in complex with known ligand RG501. Next, virtual screening of the primary focused library was performed by using automated docking program, AutoDock_Vina. After virtual screening, the final candidate compounds were selected by considering docking score and docking poses. The effects of compounds on -1 RF were tested by in vitro -1 transcription/translation coupled assay(TNT), and the -1 RF efficiencies were measured by dual luciferase assay (DLA), and SDS-PAGE.
[Results and Discussion] As a result of docking analysis, we filtered 244 of candidate compounds. Dataset was docked into the binding site of HIV-1 stem-loop. Among 244 compounds, 36 compounds were selected by docking score and visual inspection. Next, we measured the -1 RF efficiency values in the presence of candidate compounds by DLA. Three compounds (SC13, SC16, and SC32) dramatically decreased -1 RF efficiency about 60 ? 90 %. Among them, SC16 showed strong inhibition effect, and the IC50 value was 1.37 ƒÊM. In addition, we also confirmed that SC16 particularly decreased -1 RF by cell-based -1 RF assay.
[Conclusion] Through this study, we identify a small molecule ligand SC16 for HIV-1 RNA stem-loop which inhibits the -1 frameshifting in HIV-1. SC16 may serve as a lead compound to develop potential anti-frameshifting agents against HIV-1.




SKKU-6 

Macrophages play a key role in the protection by Streptococcus pneumoniae pep27 mutant vaccine
Seung Han Seon1, Sang-Yoon Choi1, David E. Briles2, Suhkneung Pyo1, Dong-Kwon Rhee1

1School of Pharmacy, Sungkyunkwan University;
2Department of Microbiology, University of Alabama at Birmingham

Streptococcus pneumoniae is responsible for high mortality, causing various invasive pneumococcal diseases. Since several problems such as serotype shifts have been found despite introduction of current pneumococcal vaccine, a new type of vaccine is needed to solve these problems. Previously, intranasal immunization of the pep27 mutant showed protection against heterologous lethal challenge indicating that the mutant could be a highly feasible vaccine candidate. To elucidate the underlying mechanism of the protection, humoral and cellular responses in immunized and non-immunized groups were compared. Although the level of IgG in the immunized group was significantly increased, there was no passive-immunity against lethal D39 challenge. Moreover, when CD4+ and CD8+ T cells were depleted from the immunized mice followed by lethal challenge, the mice did not show mortality whereas non-immunized group did indicating that T cells may not be involved in the protection. However, BALF from the immunized mice showed significantly higher level of IFN-ƒÁ when exposed to D39, indicating the involvement of IFN-ƒÁ-activated macrophages.  In in vitro study, phagocytic activities of bone marrow-derived macrophages from the immunized mice were significantly increased when exposed to D39 compared to non-immunized group. In addition, flow cytometric assay showed spleen-derived monocytes from the immunized mice differentiated much rapidly into macrophages than those from the non-immunized group. Overall, these results suggested that IFN-ƒÁ-activated macrophages, but not T and B cells, could be important for the protection from lethal infection after i.n. immunization with the pep27 mutant.




SKKU-7

 Investigation of Formulation Factors for Doxazosin Mesylate Pellets
Sang-Joon Kim, (Physical Pharmacy Lab.)

[Object] The aims of the present study were to prepare doxazosin mesylate (DXM) pellets coated by fluid-bed coater and to compare the effect of coating level, coating agents and plasticizers on its dissolution rate. 
[Methods] The doxazosin mesylate (DXM) core pellets were prepared by the extrusion-spheronization technique. The surface morphology of the core pellets and that of the coated pellets were examined by scanning electron microscopy (SEM, JSM-35CF, Jeol, Japan). To control dissolution rate of DXM from the pellets, the core pellets prepared were coated with a coating dispersions containing EudragitR RL PO/RS PO and plasticizers using a fluid-bed coater (Model D-7852, Glatt GmbH, Germany).
The dissolution rates of DXM from the pellets were measured using an USP apparatus 2 (DST-600A, Labfine Inc., Korea) at 37}0.5‹C, and stirring rate was 75 rpm. The dissolution medium was simulated gastric fluids without enzymes (pH1.2). The dissolution study of DXM pellets was performed to examine the effect of EudragitR RL PO and RS PO on sustained release. The effect of coating level on the dissolution rate was also evaluated, since it could affect directly on dissolution rate of drug. The amount of the drug released from the pellets was analyzed by using HPLC system (7000 series, Hitachi Ltd., Japan).
[Results and Discussion] The morphology of DXM pellets were examined by SEM. The core pellets were shown to have a regular spherical shape and slightly rough surface. DXM pellets were coated successfully and the coating process by fluid-bed coater was appropriate for subsequent experiments. The uniform coating layer was observed at both ratios and the coating layer got thicker as the coating layer got thicker a s the coating ratio increased. The pellets coated with PEG 6000 or TEC as a plasticizer, showed a smooth surface and the coating layer was well formed. On the other hand, the pellets coated with castor oil showed cracks on the surface.
The dissolution rate had slowed down gradually for both of EudragitR RL PO and RS PO as the coating level had increased. However, the sustained-release effect of EudragitR RS PO was much stronger than RL PO and the difference had increased distinctly as the coating level had increased. The differences of the effect on dissolution rate came from distinction of property. EudragitR RS PO had a slightly water permeable property, whereas, EudragitR RL PO had a highly permeable characteristic.
The effect of the plasticizer content ratio on the release of DXM was examined in the coated pellets with EudragitR RS PO by 2.5% coating level. The increasing concentration of plasticizers in coating solution generally shows decreased drug release profiles. The pellets coated with TEC showed the slowest dissolution rate, on the other hand, those coated with PEG 6000 showed a moderate sustained effect PEG 6000 is more soluble in water than TEC, so as less of a water soluble plasticizer, TEC, is more compatible with EudragitR RS PO which is insoluble in water. As a result, the release of DXM from pellets coated with TEC was slower than PEG 6000, so it was chosen as the plasticizer.
[Conclusion] The prepared pellets were a regular spherical shape, and showed a uniform coating layer according to the coating level. The coating thickness is a very important factor to control the release of DXM from coated pellets. The effect on a sustained-release of EudragitR RS PO is much higher, compared with EudragitR RL PO. The plasticizer also could control the release of DXM from pellets coated with EudragitR RS PO but the effect of plasticizers in EudragitR RL PO is weaker as compared to EudragitR RS PO.