10^th Asia-Pacific Pharma Congress May 08-10, 2017 Singapore

Yi-Ching Lo has her expertise in drug development and in improving the aging health. Her research interest is focusing on the development of neuroprotective and muscle enhancing agents, including chemical and natural products.

Liuwei dihuang (LWDH) is a widely used traditional Chinese medicine for neurosis, diabetics and renal disorders. Methylglyoxal (MG) is the most potent precursor of advanced glycation end products, which has been implicated in diabetic complications, cardiovascular diseases and central nervous system disorders. The present study aimed to investigate the protective effects of LWDH on MG-induced myotoxicity in C2C12 myotubes. Methods: C2C12 myoblasts were differentiated by differentiation medium to form myotube structure. C2C12 myotubes were then pre-treated with LWDH water extract (LWDH-WE) for 1 h before MG treatment. Protein expressions were analyzed by western blot analysis. Morphological changes were observed by an inverted microscope. Mitochondria membrane potential and reactive oxygen species (ROS) production were measured by flow cytometer using JC-1 staining and H2DCF-DA staining, respectively. Results: In C2C12 myotubes, LWDH-WE attenuated MG-induced reduction of mitochondrial membrane potential. Moreover, MG-induced NADPH oxidase (Nox) activation and ROS production were inhibited by LWDH-WE treatment. Furthermore, LWDH-WE attenuated MG-induced myotubes atrophy accompanied with down-regulating signaling of protein degradation pathway including Foxo3a, atrogin-1 and MuRF-1 in C2C12 cells. Conclusion. LWDH might provide protection against MG-induced myotoxicity via attenuating oxidative stress and protein degradation in C2C12 myotubes, suggesting the potential benefits of LWDH on treatment of skeletal muscle atrophy.

Tetsuo Narumi studied organometallic chemistry at Waseda University in Shinjuku, Tokyo, where he worked in the research group of Prof. Isao Shimizu. After PhD studies at Waseda University with Prof. Shimizu followed by Kyoto University with Prof. Nobutaka Fujii, he spent a year in US as a JSPS postdoctoral fellow with Prof. Jeffrey W. Bode at the University of Pennsylvania. In 2009, he began his academic career in Japan, at Tokyo Medical and Dental University with Prof. Hirokazu Tamamura. In 2013, he began his independent career at Shizuoka University, in Japan, as a associate professor in the Bioorganic Chemistry.

Naturally occurring pentacyclic triterpenoid derivatives, such as betulinic acid derivatives, have been shown to exhibit various biological activities including anti-HIV activity. IC9564 [Mayaux, JF. Et al, PNAS. 1994] and RPR103611 [Dereu, N. et al., JMC, 1996] are statin derivatives of betulinic acid tethered by 8-aminooctanoic acid linker, which was reported as a novel class of HIV-1 entry inhibitors. Although those betulinic acid derivatives show nanomolar order potency agains diverse HIV-1 strains, relatively high cytotoxicity is one of the drawbacks of them. In this study, the structure-activity relationship study of a series of triterpenoid derivatives was conducted to identify the potential triterpenoid-based HIV entry inhibitors with lower cytotoxicity than betulinic acid derivatives. Significant potency gains were made by replacing the betulinic acid moiety with the oleanoic acid, resulting in the discovery of several potent compounds. This study identified a novel lead compound OKS3-019 with significant anti-HIV activity against 89.6 strain of HIV-1 and lower cytotoxicity than those of known betulinic acid derivatives. Design, syntheses, bioevaluation and docking models of the newly identified oleanoic acid derivatives will be discussed.

Masato Yonezawa has nearly a total of ~20 years of experience in epigenetics research in academia and industries. Analyzed epigenetic phenomena by means of molecular biology and biochemistry. Highly skilled with enzymatic assays and screened several inhibitors of chromatin modifying enzymes. Would like to contribute to epigenetic research and therapy by developing useful tools and drugs.

Results from sequencing large numbers of normal vs. tumor samples from the cancer genome atlas project (TCGA) revealed a great number of potential new therapeutic targets among epigenetic enzymes and factors. An outcome of this large scale project is that there is significant interest in developing screening strategies for many of the classes of these proteins, including histone deacetylases (HDACs), histone acetyltransferases (HATs), lysine methyltransferases (KMTs), lysine demethylases (KDMs), and bromodomain containing proteins (bromodomains). While the activity of some of these targets have yet to be validated using screening platforms that use peptide substrates (i.e. Perkin Elmer AlphaLISA), Active Motif has produced a protein toolbox of reagents including active enzymes, recombinant substrates, and detection antibodies for homogenous assay platforms such as AlphaLISA. We have validated reagents used in these assays for targets HDAC3, LSD1, p300, SETDB1, and BRD family members to ensure our ability to validate these proteins for performance in screening assays. As assessed by AlphaLISA assays, the IC50 values for various reference compounds for the enzymes under evaluation, are well within published results. The protein toolbox continues to expand to include a range of enzyme substrates, from peptides and recombinant histones bearing post-translational modifications to more complex, and biologically contextual recombinant octamers, oligonucleosomes, and “designer” mononucleosomes, which include site directed post-translational modifications. The expanding portfolio of substrates will enable screens of additional therapeutic targets using these higher order nucleosome substrates which may enable identification of small molecule compounds which exhibit greater specificity or selectively in vivo relative to those currently identified using peptide substrates.

Wenhao Xia has earned his Bachelor and PhD degrees from National University of Singapore. He has worked in Takeda and GSK prior to joining PMI and has profound expertise in using preclinical in vivo models to evaluate products/compounds and to understand the mechanism of action.

Cigarette smoking is a risk factor for chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD). ApoE-/- mice are prone to developing premature atherosclerosis and emphysema making it an ideal model in which both pathologies can be assessed simultaneously. We evaluated the effects of cigarette smoke (CS) from a standard reference cigarette (3R4F) and aerosol from Tobacco Heating System 2.2 (THS2.2), a candidate modified risk tobacco product (cMRTP). ApoE-/- mice were exposed for up to 8 months to the test aerosol for 3 hours/day, 5 days/week to a target nicotine concentration of 30 µg/l. After 2 months of exposure to CS, cessation and switching groups were further exposed for up to 6 months to fresh air, or THS2.2, respectively. Multiple markers of disease progression were investigated including atherosclerotic plaque formation, pulmonary inflammation, pulmonary function and lung emphysema. Exposure to CS induced time-dependent molecular, physiological and inflammatory pulmonary responses in ApoE-/- mice consistent with emphysematous changes. The area and volume of atherosclerotic plaques measured in the aortic arches were higher in CS-exposed animals compared to both sham and cMRTP-exposed animals at all time-points. Significant changes in the lung transcriptome and proteome of ApoE-/- mice were observed in response to CS-exposure compared to sham-exposed mice. Smoking cessation and switching to THS2.2 resulted in lower activation levels compared to continuous exposure to CS. Both the cessation and switching groups showed similar effects on the histopathological and molecular endpoints, indicating significant reduced effects in comparison to the continued exposure to CS.

Sanghee Kim has expertise in the synthesis of natural/endogenous products, design and evaluation of their mimetics, and preparation of natural product-like compounds library. Over the last decade, he have been interested in lipids because they played essential roles in signal transduction, membrane trafficking and morphogenesis evaluation.

Newly isolated compounds from natural sources are important in the lead development of new therapeutics for various diseases. Currently, bioassay-guided isolation is the preferred method for identifying new natural products. However, bioassay guidance alone does not guarantee the complete exploration of natural product compounds. We believe that the systematic exploration of the natural product chemical space will be significantly accelerated by the availability of competent chemical assay systems that can reliably isolate compounds with a specific functional group. We present a new and efficient system for chemical assay-guided natural product isolation. This model system was devised for the identification and isolation of terminal alkyne-containing natural products. This new chemical assay system features a fluorogenic chemodosimeter immobilized onto a solid support. In order to isolate compounds with only the terminal alkyne functionality, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was adopted. Our newly designed sensory bead can quantitatively identify terminal alkynes on the basis of the fluorescence signal. With the guidance of our sensory chemical assay system, we were able to detect and isolate a terminal diyne from the methanol extract of Chrysanthemum morifolium. We believe that our chemical assay system is applicable in many other fields, such as metabolomics and food science.

Yun-yun Wu got master degree from Institution of Biochemistry and Molecule biology in National Yang-Ming University, learning cell-based experiment. And now studying PhD program to find hit and lead compound for ameliorating Huntington’s disease progressing. She is interested in drug development.

Huntington’s disease (HD), an inherited neurodegenerative disorder, is caused by aberrant expansion of CAG tri-nucleotide repeats in huntingtin (HTT) gene, which results in a production of mutant proteins that are detrimental to neurons. HD clinical symptoms include motor, cognitive and psychiatric disturbances, and patients usually die 10-15 years after the onset of disease. The behavior deficits and neuronal loss can be alleviated by lowering mutant HTT (mHTT) expression in a variety of model systems, suggesting mHTT is causative for genesis and progression of disease and also a target for therapeutic intervention. Despite mHTT is deleterious to neurons, the normal wild-type HTT has a neural protective role. As a result, allele-specific reduction of mHTT is a relatively favorable approach for HD treatment. Supt4h, forming a heterodimer complex with Supt5h, is a transcription elongation factor that aids RNA polymerase II during the process of transcription elongation. Earlier studies we demonstrated that disturbance of Supt4h/5h complex by lowering Supt4h protein levels results in a substantial decrease of transcript production from mHTT allele while leaving wild-type HTT allele affected marginally4. We further demonstrated that the motor function deficits of HD transgenic mice are ameliorated by Supt4h genetic knockout and that the life-span of HD mice is prolonged accordingly, suggesting Supt4h is applicable for targeting against mHTT expression and HD. Here, we designed a novel assay platform to screen small molecule compounds that are capable to interfere with the complex formation of Supt4h/5h. Among more than 200 thousands compounds tested, we identified and validated multiple hits with the nature of suppressing mHTT in mouse striatal neural cells or lymphoblastoid cells derived from HD patients. These compounds also showed a rescue effect on rough eye and declined eclosion rate that are caused by mutant HTT in HD-Drosophila models, supportive of their potential use in HD.

Seunghee Lee is working as an Associate professor in College of Pharmacy, Seoul National University.

The pathophysiologic continuum of non-alcoholic fatty liver disease begins with steatosis. Despite recent advances in our understanding of the gene regulatory program directing steatosis, how it is orchestrated at the chromatin level is unclear. PPAR2 is a hepatic steatotic transcription factor induced by overnutrition. Here, we report that the histone H3 lysine 4 methyltransferase MLL4/KMT2D directs overnutrition-induced murine steatosis via its coactivator function for PPAR2. We demonstrate that overnutrition facilitates the recruitment of MLL4 to steatotic target genes of PPAR2 and their transactivation via H3 lysine 4 methylation because PPAR2 phosphorylated by overnutrition-activated ABL1 kinase shows enhanced interaction with MLL4. We further show that Pparg2 (encoding PPAR2) is also a hepatic target gene of ABL1-PPAR2-MLL4. Consistently, inhibition of ABL1 improves the fatty liver condition of mice with overnutrition by suppressing the pro-steatotic action of MLL4. Our results uncover a murine hepatic steatosis regulatory axis consisting of ABL1-PPAR2-MLL4, which may serve as a target of anti-steatosis drug development.

Jeong hun Lee majored in Oriental pharmacy at Kyunghee University, and now studying on master course, belonging to Department of Life and Nanopharmaceutical Science, Kyunghee University. He has studied about cancer biology, especially related with apoptosis and cell cycle arrest in cancer cells and how apoptosis is induced in specific cancer cells, like lung carcinoma, by using derivatives of natural product or chemical compositions. He really wants to contribute to solving many problems about chemo resistance, one of main cause to make a difficulty to cancer’s chemotherapy.

KCP10043F(3,4-dihydroquinazoline derivative) is a selective T-type Ca2+ channel blockers and other studies showed that selective Ca2+ channel bloackers could inhibit the growth of cancer cells such as ovarian, lung, and pancreatic cancer cells. we now investigated that KCP10043F induced G1 arrest dose-dependently and cyclins and CDKs are regulated at protein expression level. In addition, KCP10043F made synergic effect on cell death with Etoposide, a novel anti-cancer agent. Then we continuously studied that KCP10043F induced apoptosis at high dose, detected by Annexin V-FITC / PI staining assay, and this cell death was dependent on caspase-activation, both capsase-3, 8 and -9. moreover, Bcl-2(anti-apoptotic protein), was down-regulated and Bax(pro-apoptotic protein) was up-regulated by this agent. This pathway was confirmed by using z-VAD-FMK, pan-caspase inhibitor, blocked the cell death induced by KCP10043F. Co-treatment with etoposide in low-dose also activated caspase-8,9. So our results suggested pathway how KCP10043F makes synergic effect on A549 cell line and it has possibility to be a potential anti-cancer agent reducing chemoresistance of lung cancers that show the lowest viability among many cancers.

Do-Hyung Kim majored in life sciences at Kyunghee University in South Korea and had experience with many natural plants in Thailand and Indonesia. He is currently a master of medicine analysis at Kyung Hee University. He has a lot of experience in drug analysis and has a lot of experience in bioequivalence experiment, clinical experiment and pharmacokinetic experiment. For example, the bioequivalence studies of oxiracetam, colchicine, and rebamipide were performed. Also, scopoletin, liquiritun and curcumin contained in natural products were analyzed by UPLC and mass spectrometry. As a result, it contributed to the development of new drugs and generic drugs, and plans to expand the scope of development of new drugs and biopharmaceuticals.

A rapid, specific and fully validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Fimasartan and amlodipine using BR-A-563 and clarithromycin as an internal standard, respectively. Liquid-liquid extraction (LLE) was carried out on 0.05 mL of human plasma using ethyl acetate and hexane for Fimasartan and 0.2 mL of human plasma using methyl tert-butyl ether (MTBE) and methyl chloride (MC) for amlodipine. Detection was performed in positive ion multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 502.4 → 207.1 for Fimasartan, m/z 526.48 → 207.2 for BR-A-563, m/z 408.9 → 238.0 for amlodipine and m/z 748.2 → 158.0 for clarithromycin, respectively. Chromatographic separation was performed on Kinetex C18 (75 × 2.1 mm, 2.6 µm) using a mobile phase consisting of 0.05% formic acid- methanol (30:70, v/v) at a flow rate of 0.2 mL/min for Fimasartan and Luna C18 (50 × 2.0 mm, 3.0 µm) using a mobile phase consisting of 0.1% acetic acid-methanol (30:70, v/v) at a flow rate of 0.2 mL/min for amlodipine. The linear calibration curves were 1-500 ng/mL for Fimasartan and 0.2-20 ng/mL for amlodipine. The total runtime was 2.5 min for Fimasartan and 3 min for amlodipine, retention time was about 1.6 and 1.0 min for Fimasartan and amlodipine, respectively. The intra-day and inter-day reproducibility was less than 12% for each analyte. The proposed method shows good separation of analytes, without interference from endogenous substances. Fimasartan and amlodipine were found to be stable under these conditions and the method was successfully applied to the pharmacokinetic study of complex tablet (Dukarb®, 60 mg Fimasartan and 10 mg Amlodipine).

Seung-Bin Lee has been intensively studied on screening anti-inflammatory effect among various natural product derived compounds. We encounter incalculable kinds of inflammatory situations, starting from tiny skin abrasion up to septic shocks. Not only physical inflammatory diseases such as inflammatory-bowel diseases and rheumatoid arthritis, but also stress-induced inflammations are being increased throughout decades. In an idea to alleviate these tendency, he has been investigated the underlying molecular mechanism of several drugs which elicit significant decrease of inflammatory endpoints such as nitric oxide, prostaglandin E2, and pro-inflammtory cytokines. It could be meaningful so far to seek out such proper drugs to prevent unpleasant encounter on pro-inflammatory states.

Xanthotoxin has been reported to possess skin-protective and anti-oxidative properties. However, anti-inflammatory property has not been studied to date. Therefore, we investigated the role that xanthotoxin plays on anti-inflammatory activity as well as its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. In LPS-induced macrophages, xanthotoxin was found to inhibit nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) in a concentration-dependent manner. It also suppressed the LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression at the protein level and iNOS, COX-2, TNF-α, and IL-6 at the mRNA level. Molecular mechanism shows that xanthotoxin attenuated the LPS-induced transcriptional and DNA-binding activity of activator protein-1 (AP-1), and this was associated with a decrease in the phosphorylation of c-Fos instead of c-Jun. It played a suppressive effect on the transcriptional and DNA-binding activity of nuclear transcription factor kappa-B (NF-κB) through the inhibiting of p65 nuclear translocation. In addition, the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) was found to be suppressed by xanthotoxin. Taken together, these results indicate that xanthotoxin decreased NO, PGE2, TNF-α, and IL-6 production through downregulation of NF-κB, AP-1, and JAK/STAT signaling pathways in LPS-induced RAW 264.7 macrophages.

Saravanan Govindaraju is a PhD student in Prof.Yun’s lab, dept of Bionanotechnology, Gachon University, South Korea. He graduated Masters degree from the same university and bachelor degree from Anna University, India. His research experience includes synthesis of nanomaterials, cell cytotoxicity study and bio-AFM imaging.

Fluorescent gold cluster (AuNCs) have gained much attention due to their wide spread property like fluorescence, small size, non-toxic, stability and can be easily conjugated to the bio molecules. Curcumin (CUR), a polyphenol compound derived from Curcuma longa plant, is a promising anticancer agent for various tumors. We hypothesized the synthesis of fluorescent gold cluster with the conjugation of curcumin for the evaluation of biophysics, biomechanics and cytotoxicity study in cancer cell. Synthesized nanomaterial gives strong red photoluminescence at 650 nm and FTIR confirms the conjugation of CUR to the AuNCs. HRTEM analysis divulges size range from 4-6 nm of monodispersed particles. CUR-AuNCs didn’t show any toxicity to the human fibroblast cell line and more toxic to HeLa cells. Optical fluorescence microscopes exhibited that CUR-AuNCs killed specifically to cancer cells. Bio-AFM image provide the morphological changes of HeLa cells after the treatment of CUR-AuNCs at different time interval. In future, we extent our research to biophysics and biomechanics study of CUR-AuNCs by Bio-AFM, and the cluster treated for the anticancer effect in xenograft model.

Hsien-Yi Chiu is an attending physician of dermatology department and research scientist at the Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University. He has contributed significantly to a number of important studies on immune-related skin disorders, including urticarial and psoriasis.

Approximately 20~30% of the general population experiences at least one episode of urticaria in their lifetime.1,2 Research has also shown that itch, the dominant symptom in most patients with acute urticaria, severely interferes with sleep and daily activities, and has a detrimental impact on the quality of life. 1,2,3 This prospective, interventional, single-arm open –label trial recruited 20 consecutive patients aged 20-75 years with a diagnosis of acute urticaria persistant (wheals between 3 days ~ 6 weeks) and baseline urticaria activity score (UAS)  4 despite oral/intravenous antihistamines with or without systemic corticosteroid therapy who attended our clinic between January 2015 and October 2015. At day 0, patients received a single dose of 300 mg of omalizumab subcutaneously.4,5 Treatment with omalizumab resulted in mean UAS decrease from 5.0 ± 0.8 at baseline to 1.6 ± 2.1 at day 7. Compared with baseline, a statistically significant reduction in UAS had been observed since day 1, and continued through week 6 (Figure 1). Ten patients (50%) achieved complete remission (UAS= 0 or UCT=16) at day 7 of omalizumab therapy . As with the primary efficacy outcome, similar improvements were also observed for ISS, UCT, and DLQI (Figure 1). The mean change from baseline in ISS was -0.15, -0.8, -1.30, -1.35, -1.45 at days 1, 3, 5, 7 and week 6, respectively. Mean DLQI score also decreased by 16.4 points at day 7 after omalizumab treatment. Similar with UAS, sustained therapeutic improvement in ISS, UCT, and DLQ was also observed till week 6. The mean of the patients’ percentage improvements in UCT and DLQI was 75.1% and 55.6 % at day 7, respectively. Our study demonstrated that despite significantly improved symptoms and quality of life in patients with acute urticaria after omalizumab treatment, a rapid control was observed in a minority.

Initially trained as a cellular and molecular biologist, Blaine began his scientific career studying the potential of differentiated embryonic stem cells as cellular replacement therapies in a biotechnology start-up company. This led to a journey spanning 3 continents working in the pharmaceutical and the tobacco industries in research and development departments focusing on assay development and drug discovery, as well as inhalation toxicology.

CHTP (carbon heated tobacco product) 1.2 is a potential modified risk tobacco product in which the tobacco plug in a specially designed stick is heated to ≤ 300°C using a carbon heat source. The operating temperature is below the combustion temperature of tobacco, resulting in generation of aerosol with significant reduction of harmful and potentially harmful constituents compared with cigarette smoke. The toxicity of CHTP 1.2 was characterized in a 90-day sub-chronic inhalation study according to the OECD 413 testing guidelines. Sprague-Dawley rats were exposed for 6 hours per day, 5 days per week for at least 13 weeks to filtered air (control), mainstream smoke of reference cigarette 3R4F at 23 µg nicotine/L, or aerosol of CHTP 1.2 at three target concentrations of 15, 23 and 50 µg nicotine/L, respectively. Additional animals from control and CHTP 1.2 high exposed groups were included to observe reversibility of toxicity over a period of 42 days after the exposure. Reduction in respiratory minute volume and frequency typically observed in 3R4F-exposed group was less pronounced in animals breathing CHTP 1.2 aerosol. The number of inflammatory cells and levels of excreted pro-inflammatory cytokines in bronchoalveolar lavage fluid of animals exposed to CHTP 1.2 were lower than in the 3R4F-exposed group. Clinical pathological changes such as higher blood neutrophil counts, elevated liver enzymes and decrease of cholesterol and glucose levels were observed in 3R4F and lesser extent in CHTP 1.2 high groups, compared with control. Microscopic findings in respiratory tract organs including epithelial cell hyperplasia and squamous metaplasia were reduced in CHTP 1.2 as compared with 3R4F-exposed group. In summary, the results indicate that the inhalation of aerosol from CHTP 1.2 caused minor effects in rats mainly attributed to nicotine, and the effects on respiratory tract organs were much lower compared with those from 3R4F reference cigarette.