Book of Abstracts - IAPC · 2018. 10. 21. · Elrashid Saleh Mahdi, Nawal Elhadi, Noon Jar Elnabi, Gamal Khalaf Allah Mohd Ali _ 35 The Ultrafine Formulation of Betulin with Biocompatible

5th

IAPC Meeting:

Emerging Technologies in

Drug Discovery and

Development

Programme

&

Book of Abstracts

Zhuhai, PR China, August 23-26, 2016

5th IAPC Meeting: Emerging Technologies in Drug Discovery and Development 5th World Conference of Physico-Chemical Methods in Drug Discovery and Development & 2nd World Conference on ADMET and DMPK Zhuhai, PR China, August 23-26, 2016

Programme & Book of Abstracts Published by

International Association of Physical Chemists E-mail: [email protected], URL: http://www.iapchem.org For Publisher Zoran Mandić Editors Zoran Mandić & Kin Tam Design, Page Making and Computer Layout Aleksandar Dekanski Circulation 150 Copies

mailto:[email protected]

http://www.iapchem.org/

The Scientific and Organizing Committee:

Alex Avdeef, in-ADME Research, USA

Elena Boldyreva, Russian Academy of Sciences, Russia

Biserka Cetina-Čižmek, PLIVA, Croatia

Chi-Chung Chan, WuXi AppTec (Shanghai) Co. Ltd, China

Christopher Chan, Industrial Technology Research Institute, Taiwan

Weiguo Dai, Johnson and Johnson, USA

Rolf Hilfiker, Solvias, Switzerland

Josef Jampilek, University of Veterinary and Pharmaceutical Sciences,

Czech Republic

Thomas Leung, Hong Kong Polytechnic University, Hong Kong

Qingfei Liu, Tsinghua University, China

Zoran Mandić, University of Zagreb, Croatia - conference co-chair

Christos Reppas, University of Athens, Greece

Marti Rosés, University of Barcelona, Spain

Abu Serajuddin, St. John's University, USA

Joong Sup Shim, University of Macau, Macau

Kiyohiko Sugano, Toho University, Japan

Krisztina Takács-Novák, Semmelweis University, Hungary

Kin Tam, University of Macau, Macau - conference co-chair

Yin Tian, Chongqing University of Posts and Telecommunications, China

Henry Tong, Macao Polytechnic Institute, Macau

Klara Valko, GSK, UK

Tatjana Verbić, University of Belgrade, Serbia

Hong Wan, Shanghai Hengrui Pharmaceutical, China

v

CONTENTS

CONFERENCE PROGRAMME ______________________________________________ IX

ORAL PRESENTATIONS __________________________________________________ 1

Finding Needles in Very Large Haystacks: DNA-Encoded Libraries (Dels) for Lead Generation

Jin Li _________________________________________________________________ 3

Role of Physico-Chemical and ADME Properties in Cancer Drug Development Godefridus J. Peters _____________________________________________________ 4

Development of Fluorescent Biosensors for Screening of Inhibitors of Some Bacterial Enzymes

Kwok-Yin Wong ________________________________________________________ 5

Development of Drug Nanocrystals for Theranostics Wei Gao, Tonglei Li _____________________________________________________ 6

A Small Scale Method to Determine Release Rate from Complex Carrier-Mediated Systems

Caroline Alvebratt, Erik Petersson, Maria Strömme, Christel A. S. Bergström ________ 7

Impact of Polymorphism on the Development and Manufacturing of Pharmaceuticals Rolf Hilfiker ___________________________________________________________ 8

Cocrystal Solubility-pH Data Analysis using pDISOL-X (with Automatic Equilibrium Equation Generation)

Alex Avdeef ___________________________________________________________ 9

Two Novel Cocrystals with Improved Solubility and in Situ Monitoring of Cocrystallization of Lamotrigine

Shichao Du, Yan Wang, Jingkang Wang, Junbo Gong _________________________ 10

Drugs as Materials: Processing, Structure, Properties Elena Boldyreva _______________________________________________________ 11

The Importance of Proton Transfer in Amorphous Active Pharmaceutical Ingredients Marzena Rams-Baron, Żaneta Wojnarowska, Mateusz Dulski, Patryk Włodarczyk, Marian Paluch ________________________________________________________ 12

Mechanism-Based Selection of Stabilization Strategy for Amorphous Formulation Khadijah Edueng, Denny Mahlin, Christel Bergström __________________________ 13

Discovery and Development of Pyrotinib: A Novel Irreversible EGFR/HER2 Dual Tyrosine Kinase Inhibitor with Favorable Safety Profiles for the Treatment of Breast Cancer

Xin Li, Changyong Yang, Hong Wan, Ge Zhang, Jun Feng, Lei Zhang, Xiaoyan Chen, Dafang Zhong, Liguang Lou, Weikang Tao, Lianshan Zhang ____________________ 14


vi

Epiberberine, a Natural Protoberberine Alkaloid, Inhibit Urease of Helicobacter pylori and Jack Bean via Binding to the Sulfhydryl Groups

Lihua Tan, Cailan Li, Hanbin Chen, Zhizhun Mo, Jiangtao Zhou, Yuhong Liu, Zhilin Ma, Yuyao Xu, Xiaobo Yang, Jianhui Xie, Ziren Su ________________________________ 15

Phenyl Butyrate Inhibits Pyruvate Dehydrogenase Kinase 1 and Contributes to its Anti-Cancer Effects

Wen Zhang, Xiaohui Hu, Shao-Lin Zhang, Kin Yip Tam _________________________ 16

A Potential Target for Parkinsonism Treatment Tao Chen, Bo Jiang, Yuan Xu, Feng Zhi, Yiling Yang, Ying Xia ____________________ 17

Excessive Porphyrin Production Induced by Cyclohexylmethyl Flavonoids Suppresses Self-Renewal Propagation of Breast Cancer Stem Cells

Wen-Ying Liao, Chih-Chuang Liaw, Chien-Shu Chen, Sheng-Chu Kuo, Yuan-Chao Huang, Chia-Ning Shen ________________________________________ 18

Predictors for Drug Effects with Alzheimer’s Disease: Shed New Light from EEG Parameters to Brain Connectomics

Li Yang, Zhongyan Wang, Huiling Zhang, Wei Xu, Shuxing Zheng, Haiyong Zhang, Yin Tian _____________________________________________________________ 19

Data Mining for Equilibrium Solubility of Ionizable Druglike Molecules Alex Avdeef __________________________________________________________ 20

How to Optimize the Physicochemical and Biomimetic Properties of Putative Drug Molecules?

Klara Valko ___________________________________________________________ 21

Comparison and Modelling of Experimental and Calculated Distribution Coefficient (Logd) Values at Various pHs

Mare Oja, Uko Maran __________________________________________________ 22

Quantitative Structure Activity Relationship (QSAR) Analysis of Indirubin Derivatives as Cyclin Dependent Kinase 5 (CDK5) Inhibitor

Muhammad Arba, Rahmad Sutrisna, Ruslin _________________________________ 23

Design and Molecular Docking Study of Novel Indirubin Derivatives As Inhibitor of Cyclin Dependent Kinase 5 (CDK5)

Ruslin, Rahmad Sutrisna, Muhammad Arba _________________________________ 24

Strategies for the Development of Liquid, Semisolid and Solid Lipid-Based Drug Delivery Systems

Abu T. M. Serajuddin ___________________________________________________ 25

Study on Effective Methods for Stabilizing the Amorphous Forms of Anti-Cholesterol Drug: Ezetimibe

Marian Paluch, Justyna Knapik ___________________________________________ 26

Theoretical Investigation on Bioequivalence and Biowaiver Scheme Kiyohiko (Kiyo) Sugano _________________________________________________ 27

5th IAPC Meeting: Emerging Technologies in Drug Discovery and Development

vii

Anti-Tumour Effects of a Human & Mouse Cross-Reactive’ Anti-TACE Antibody in a Pancreatic Cancer Model In Vivo

Shun Ming Yuen, Jie Ye, Gillian Murphy, Ruiyu Xie, Hang Fai Kwok _______________ 28

POSTER PRESENTATIONS _______________________________________________ 29

Design, Synthesis and Antitumor Activity of IMB-1506 Derivatives Jun Zhang, Xiaoning Li __________________________________________________ 31

Design, Synthesis and In vitro Antitumor Activity of Vandetanib Derivatives Xiaoning Li, Jun Zhang __________________________________________________ 32

Geniposide Improves Function and Reduces Apoptosis in High Glucose-induced Glucotoxic Insulinoma Cells

Lixia Guo, Xuxu Zheng, Jianhui Liu, Zhongyi Yin ______________________________ 33

DSC as an Identification Tool for Protein Drugs and Protein Test Systems Jamlet Monaselidze, Mikheil Gadabadze, Maya Gorgoshidze, Maya Kiladze, David Khachidze, Shota Barbakadze ____________________________________________ 34

Assessment of the Impact of Distribution and Storage Condition on the Quality of Medicines Marketed by National Medical Supplies Fund in Sudan Healthcare Network

Elrashid Saleh Mahdi, Nawal Elhadi, Noon Jar Elnabi, Gamal Khalaf Allah Mohd Ali _ 35

The Ultrafine Formulation of Betulin with Biocompatible Carriers Exhibiting Improved Dynamics of Dissolution

Svetlana A. Myz, Andrey G. Ogienko, Anna A. Ogienko, Tatyana P. Shakhtshneider, Svetlana A. Kuznetsova _________________________________________________ 36

The Approaches to Preparation of Betulin Esters Composites with Improved Biological Activity

Tatyana P. Shakhtshneider, Yuriy N. Malyar, Mikhail A. Mikhailenko, Anna S. Zamay, Natalia A. Pankrushina, Svetlana A. Kuznetsova ________________ 37

Screening of Polysaccharides from Marine Microalgae and Study on its Antioxidant Activity

Zhan Jingting, Huang Yongmei, Yang Xiaohong, Luo Hui, Jiang Liming ____________ 38

The Extraction and Determination of Fatty Acids of Marine Microalgaes from Naozhou Island

Huang Yongmei, Yang Xiaohong, Zhan Jingting, Xue Shanshan, Luo Hui, Jiang Liming 39

A New Polymorph of Metacetamol – Promising Analogue of Paracetamol Denis A. Rychkov, Lindsay McGregor, Paul L. Coster, Sarah Day, Valeri A. Drebushchak, Andrei F. Achkasov, Gary S. Nichol, Colin R. Pulham, Elena V. Boldyreva _____________________________________________________ 40

Conformational Polymorphs of Furosemide Solvates Alina A. Beloborodova, Vasily S. Minkov, Denis A. Rychkov, Elena V. Boldyreva _____ 41

Simple and Efficient Modifications of Well Known Techniques for Reliable Growth of High-Quality Crystals of Small Bioorganic Molecules

Sergey G. Arkhipov, Denis A. Rychkov, Elena V. Boldyreva ______________________ 42


viii

The Glucuronidated and Sulfated Metabolites of Tilianin are Substrates of Breast Cancer Resistant Protein (ABCG2) in Mice

Qingwei Chen, Liping Wang, Xuejun Zeng, Qiang Li, Lijun Zhu, Ming Hu, Zhongqiu Liu, Xinchun Wang _____________________________________________ 43

Synthesis and Validation of (R)-3,3,3-trifluoro-2-hydroxy-2-Methylpropionamides as Pyruvate Dehydrogenase Kinase Inhibitors To Reduce the Growth of Cancer Cells

Shao-Lin Zhang, Wen Zhang, Zheng Yang, Xiaohui Hu, Kin Yip Tam ______________ 44

Screening for Culturable Marine Bacteria Producing Exopolysaccharide from the Intertidal Zone of Zhanjiang Coast

Shan-Shan Xue, Ying-Wen Liu, Li-Jiao Cui, Yong-Mei Huang, Li-Ming Jiang _________ 45

Study on Screening of Oil-Producing Bacteria in Five Methods Ying-Wen Liu, Shan-Shan Xue, Li-Jiao Cui, Yong-Mei Huang, Li-Ming Jiang _________ 46

SLM Attenuates Apoptosis Induced by Amyloid Beta 1-42 in SH-SY5Y Human Neuroblastoma Cells

Xiaoli Wu, Jayasankar Kosaraju, Xiaohui Hu, Kin Yip Tam ______________________ 47

The Effects of Icariin on the Macrophage Apoptosis Induced by Ox-LDL and the Underlying Mechanisms

Luo Juan, Ji Yanqiong, Tan Yan, Pan Longrui, Wang Xuanbin, Li Hongliang, Zhang Qiufang ________________________________________________________ 48

Tanezumab for Patients with Osteoarthritis of the Knee: A Meta-Analysis Shun-Li Kan, Yan Li, Guang-Zhi Ning, Ling-Xiao Chen, Shi-Qing Feng ______________ 49

AUTHOR INDEX ________________________________________________________ 50

Conference Programme


xi

Monday, August 22, 2016 15:00 – 19:00 Participant registration / Poster mounting 19:30 – Welcome party – Dehan hotel

Tuesday, August 23, 2016 08:00 – 08:30 Participant registration / Poster mounting 08:30 – 08:45 Opening ceremony

Session 1. Recent advances in the physico-chemical/activity profiling of drug substances Chairperson Alex Avdeef 08:45 – 09:30

OP 01 Jin Li, Hitgen Ltd., China Finding Needles in Very Large Haystacks: DNA-Encoded Libraries (Dels) for Lead Generation

09:30 – 10:15 OP 02

Godefridus J. Peters, VU University Medical Center, The Netherlands Role of Physico-Chemical and ADME Properties in Cancer Drug development

10:15 – 10:45 Coffee break / Poster session Chairperson Godefridus Peters 10:45 – 11:30

OP 03 Kwok-Yin Wong, The Hong Kong Polytechnic University, China Development of Fluorescent Biosensors for Screening of Inhibitors of Some Bacterial Enzymes

11:30 – 12:15 OP 04

Wei Gao, Tonglei Li, Purdue University, USA Development of Drug Nanocrystals for Theranostics

12:15 – 12:40 OP 05

Caroline Alvebratt, Erik Petersson, Maria Strömme, Christel A.S. Bergström, Uppsala University, Sweden A Small Scale Method to Determine Release Rate from Complex Carrier-Mediated Systems

Lunch Session 2. Recent advances in solid state characterization Chairperson Elena Boldyreva 14:30 – 15:15

OP 06 Rolf Hilfiker, Solvias AG, Switerland

Impact of Polymorphism on the Development and Manufacturing of Pharmaceuticals

15:15 – 16:00 OP 07

Alex Avdeef, in-ADME Research, USA Cocrystal Solubility-pH Data Analysis using pDISOL-X (with Automatic Equilibrium Equation Generation)

16:00 – 16:20 OP 08

Shichao Du, Yan Wang, Jingkang Wang, Junbo Gong, Tianjin University, China Two Novel Cocrystals with Improved Solubility and In Situ Monitoring of Cocrystallization of Lamotrigine

16:20 – 16:50 Coffee break / Poster session

Chairperson Rolf Hilfiker 16:50 – 17:35

OP 09 Elena Boldyreva, Novosibirsk State University, Russia Drugs as Materials: Processing, Structure, Properties

17:35 – 18:05 OP 10

Marzena Rams-Baron, Żaneta Wojnarowska, Mateusz Dulski, Patryk Włodarczyk, Marian Paluch, University of Silesia, Poland The Importance of Proton Transfer in Amorphous Active Pharmaceutical Ingredients

18:05 – 18:30 OP 11

Khadijah Edueng; Denny Mahlin; Christel Bergström, Uppsala University, Sweden Mechanism-Based Selection of Stabilization Strategy for Amorphous Formulation

19:30 - Conference dinner / Dehan hotel


xii

Wednesday, August 24, 2016

Session 3. Recent advances in drug discovery

Chairperson Chia-Ning Shen

08:30 – 09:15 OP 12

Hong Wan, Shanghai Hengrui Pharmaceutical CO., Ltd, China Discovery and Development of Pyrotinib: A Novel Irreversible EGFR/HER2 Dual Tyrosine Kinase Inhibitor with Favorable Safety Profiles for the Treatment of Breast Cancer

09:15 – 09:45 OP 13

Lihua Tan, Cailan Li, Hanbin Chen, Zhizhun Mo, Jiangtao Zhou, Yuhong Liu, Zhilin Ma, Yuyao Xu, Xiaobo Yang, and Jianhui Xie, Ziren Su, Guangzhou

University of Chinese Medicine, China Epiberberine, a Natural Protoberberine Alkaloid, Inhibit Urease of Helicobacter pylori and Jack Bean via Binding to the Sulfhydryl Groups

09:45 – 10:15 OP 14

Wen Zhang, Xiaohui Hu, Shao-Lin Zhang, Kin Yip Tam, University of Macau



Chairperson Hong Wan

10:45 – 11:30 OP 15

Tao Chen, Bo Jiang, Yuan Xu, Feng Zhi, Yiling Yang, Ying Xia, The University of Texas McGovern Medical School, USA A Potential Target for Parkinsonism Treatment

11:30 – 12:15 OP 16

Wen-Ying Liao, Chih-Chuang Liaw, Chien-Shu Chen, Sheng-Chu Kuo, Yuan-Chao Huang, and Chia-Ning Shen, Genomics Research Center, Academia

Sinica, Taiwan Excessive Porphyrin Production Induced by Cyclohexylmethyl Flavonoids Suppresses Self-Renewal Propagation of Breast Cancer Stem Cells

12:15 – 12:40 OP 17

Li Yang, Zhongyan Wang, Huiling Zhang, Wei Xu, Shuxing Zheng, Haiyong Zhang, Yin Tian, Chong Qing, University of Posts and Telecommunications, China Predictors for drug effects with Alzheimer’s disease: shed new light from EEG parameters to brain connectomics

Lunch

Session 4. Recent advances in computational methods/xiiodelling, biophysical and ana-lytical techniques of importance in medicinal chemistry and pharmaceutical research Chairperson Abu Serajuddin

14:30 – 15:15 OP 18

Alex Avdeef, in-ADME Research, USA Data Mining for Equilibrium Solubility of Ionizable Druglike Molecules

15:15 – 16:00 OP 19

Klara Valko, Bio-Mimetic Chromatography, UK How to Optimize the Physicochemical and Biomimetic Properties of Putative Drug Molecules?

16:00 – 16:30 OP 20

Mare Oja, Uko Maran, University of Tartu, Estonia Comparison and Modelling of Experimental and Calculated Distribution Coefficient (Logd) Values at Various pHs



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Chairperson Kiyohiko Sugano 17:00 – 17:35

OP 21 Muhammad Arba, Rahmad Sutrisna, Ruslin, Universitas Halu Oleo, Indonesia

Quantitative Structure Activity Relationship (QSAR) Analysis of Indirubin DerivativesaAs Cyclin Dependent Kinase 5 (CDK5) Inhibitor

17:35 – 18:10 OP 22

Ruslin, Rahmad Sutrisna, Muhammad Arba, Universitas Halu Oleo, Indonesia

Design and Molecular Docking Study of Novel Indirubin Derivatives as Inhibitor of Cyclin Dependent Kinase 5 (CDK5)

18:10 – 18:30 Poster dismounting

Thursday, August 25, 2016

Session 5. Recent advances in drug development Chairperson Tonglei Li

09:00 – 09:45 OP 23

Abu T. M. Serajuddin, St. John’s University, USA Strategies for the Development of Liquid, Semisolid and Solid Lipid-Based Drug Delivery Systems

09:45 – 10:30 OP 24

Marian Paluch and Justyna Knapik, University of Silesia, Poland Study on Effective Methods for Stabilizing the Amorphous Forms of Anti-Cholesterol Drug: Ezetimibe

10:30 – 11:00 Coffee break

Chairperson Klara Valko

11:00 – 11:45 OP 25

Kiyhiko Sugano, Toho University, Japan Theoretical Investigation on Bioequivalence and Biowaiver Scheme

11:45 – 12:30 OP 26

Shun Ming Yuen, Jie Ye, Gillian Murphy, Ruiyu Xie and Hang Fai Kwok, University of Macau, Macau Anti-Tumour Effects of a Human and Mouse Cross-Reactive’ Anti-TACE Antibody in a Pancreatic Cancer Model In Vivo

12:30 Conference closing

14:00 Optional excursion: Zhuhai sightseeing tour and dinner

Oral Presentations


Page 3

Finding Needles in Very Large Haystacks: DNA-Encoded Libraries (Dels) for Lead Generation

Jin Li

HitGen, Ltd Innovative drug discovery research activities are known for big investment, high risk and long development time as well as facing other challenges. Lead generation is the starting point and one of the most important steps during the early phase of drug discovery R&D, which requires large compound library and advanced screening technology and can only be accomplished by some major international pharmaceutical companies with abundance of capital and cutting-edge technology. The emergence of vast DNA Encoded Library has gradually filled the gap in this field and decreaseed the technological barriers for Chinese drug companies in the hit identification. As the integration of Combinatorial Chemistry and High Throughput Sequencing, it not only improves small molecule library to 100Mn even 10Bn, but also makes compound screening more convenient and effective. The efficient application of DNA Encoded Library will play a key role in the drug discovery and become a really important source for innovative drug discovery.

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Role of Physico-Chemical and ADME Properties in Cancer Drug Development

Godefridus J. Peters

Dept. of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands

Development of cancer drugs has changed considerably in the last few decades, by designing drugs that inhibit essential signaling pathways. Cancer cells are often addicted to one specific pathway and inhibition of this pathway is usually lethal. Normal cells are dependent on several signaling pathways, which compensate each other when one is inhibited. Since this novel class of drugs inhibits such a specific target in cancer cells, these drugs are often called molecular targeted drugs. These drugs were considered to be less toxic than the classical cytotoxic chemotherapeutic drugs, but it is now clear that these drugs are often as toxic as the classical drugs. However, toxicity usually has a different pattern and is more complicated to treat. Targeted drugs can be divided in large molecules (antibodies directed against specific membrane receptors) and relatively small molecules (MW <500 D), that target the active (often membrane-associated) site of these receptors, usually the ATP binding site of the protein kinase. Phosphorylation of these kinases initiates a cascade of signaling reactions, often leading to induction of cell growth. Initial development of protein kinase inhibitors often neglected basal physico-chemical and ADME properties. The drugs did not reach their target because of poor uptake in the gut, a rapid first pass effect, rapid degradation, rapid cellular efflux, or trapping in vesicles and organelles. Proper evaluation of physico-chemical properties and the use of appropriate ADME models has improved the success rate. Many of these drugs have a poor solubility, but a proper formulation often leads to an adequate passage of the gut. Moreover, passage of the gut can be improved by using their protein binding properties and administer the drug with food, which will improve uptake and prevent elimination via the gut, (e.g. lapatinib). A poor intestinal passage can be predicted with the CaCo2 system and cellular uptake with solid tumor cell lines. The combination of various models demonstrated that dasatinib has a poor uptake by organic cation transporters (OCT) and is rapidly being effluxed by one of the ABC transporters, which makes it inactive for solid tumors, but the drug will reach a sufficiently high plasma level to attack hematological disorders. Several drugs will selectively accumulate in the skin, resulting in discoloration (sunitinib) and often rash (erlotinib, gefitinib). Resistance can occur by rapid Phase I or II metabolism (e.g. sorafenib by glucuronidation, imatinib by oxidation), or accumulation of protonated species in lysosomes (e.g. sunitinib, crizotinib). Early recognition of these drug properties will prevent that potentially active drugs (or class of drugs) which would fail because of poor physico-chemical properties, can still be developed. The use of appropriate ADME models can predict a poor transport or rapid metabolism. Such results will lead to the re-design of novel analogs of the lead compounds with better properties

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Page 5

Development of Fluorescent Biosensors for Screening of Inhibitors of Some Bacterial Enzymes

Kwok-Yin Wong

Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University,

Hunghom, Kowloon, Hong Kong, China

Many inhibitors of bacterial enzymes are antibiotics, the most well known examples being

-lactams for DD-transpeptidases and clavulanic acid for -lactamases. With the emergence of antibiotic resistant bacterial strains like Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococci, Carbapenem-Resistant Enterobacteriaceae and MCR-1 containing E. coli, there is a definite need to develop new antibiotics based on new

targets and novel mechanisms. Bacterial enzymes such as -lactamases (which destroy the

-lactam antibiotics through hydrolysis of the -lactam ring) and peptidoglycan glycosyltransferases (which are responsible for polymerization of lipid II in bacterial cell wall synthesis) are important drug targets in the development of new antibiotics. In the search for new inhibitors, compound library are often screened based on various methodologies. In this presentation, we will discuss our work on the development of fluorescent biosensors for inhibitor screening. Our approach is to turn the bacterial enzyme target into a biosensor in which a fluorescent signal is given out when an inhibitor binds to the drug

target. Two classes of important targets, namely -lactamases and peptidoglycan glycosyltransferases (PGT) will be discussed. Enzyme mutants containing tryptophan on an appropriate location of the enzyme were constructed. A fluorescein label, the fluorescence of which can be quenched by tryptophan through photo-induced electron transfer mechanism, is introduced either onto the enzyme or on a known inhibitor of the enzyme. When an inhibitor enters the active site of the enzyme, the fluorescein molecule was displaced from its orginal location if it is labelled on the enzyme itself, or displaced from the active site through competitive binding if it is labelled on a known inhibitor. The quenching effect was then released and the fluorescence of the fluorescein label was restored.

The use of the above biosensors in screening of inhibitors for -lactamases and PGT will be discussed in the presentation. Acknowledgment: This work was supported by the State Key Laboratory fund from the Innovation and Technology Commission of Hong Kong and The Hong Kong Polytechnic University.

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Development of Drug Nanocrystals for Theranostics

Wei Gao, Tonglei Li

Department of Industrial & Physical Pharmacy, Purdue University, West Lafayette, Indiana, 47907, USA

Our studies of developing drug nanocrystals have progressed over the last few years with a focus on using hybrid nanocrystals for simultanenous delivery of therapeutic and bioimaging agents. The innovation idea of hybrid nanocrystal is to physically integrate guest substances directly into the crystal lattice of drug nanocrystals, as demonstrated in our studies of hybrid nanocrystals [1-3]. The idea is inspired by a common phenomenon in solid-state chemistry wherein impurities or guest chemicals can be physically entrapped in a host crystal. The existence of foreign substances in a host is typically insignificant, having little effect on the structure and integrity of the hosting crystal structure but capable of casting drastic influence on the optical appearance and other physical properties of the host. Examples are abundant, ranging from minerals (e.g., pink diamonds) to metallic (e.g., alloys) and to semiconductors (e.g., n- and p-type silicon). Preparation of guest/dye inclusion crystals is straightforward. As long as the guest substance is present in the growth medium, the guest molecules or atoms will be integrated into the crystal lattice of a host. The faster the growth rate, the more the guest can be included due to thermodynamics of crystallization. Because the nanocrystals are produced by swift precipitation, the integration ratio is commonly between 0.1 and 1 % of host crystals (weight/weight ratio). For bioimaging purposes, such ratio is sufficient, as demonstrated in our previous studies. Because a majority of anticancer drugs are poorly soluble in water, formulating them into nanocrystals circumvents using carrier or encasuplating chemicals, overcoming side effects that may be introduced by the excipients. Moreover, nanocrystals are generally more stable than other delivery systems. By integrating near infrared (NIR) dyes into nanocrystals of a chemotherapeutic compound, such as paclitaxel, we have discovered that not only these nanocrystals exhibited similar and better antitumor effects compared with conventional formulations, but these hybrid nanocrystals also permited real-time, whole-body imaging [1-3]. Our current efforts focus on using specialized dyes that may differentiate physical states of the drug in vivo, being crystalline or dissolved. [1] R.S. Zhao, C.P. Hollis, H. Zhang, L.L. Sun, R.A. Gemeinhart, T.L. Li, Molecular Pharmaceutics 8

(2011) 1985-1991. [2] C.P. Hollis, H.L. Weiss, B.M. Evers, R.A. Gemeinhart, T. Li, Pharmaceutical Research 31 (2013)

1450-1459. [3] C.P. Hollis, H.L. Weiss, M. Leggas, B.M. Evers, R.A. Gemeinhart, T. Li, Journal of Controlled

Release 172 (2013) 12-21.

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A Small Scale Method to Determine Release Rate from Complex Carrier-Mediated Systems

Caroline Alvebratt, Erik Petersson, Maria Strömme, Christel A. S. Bergström

Uppsala University, Sweden

To address the limitations of poorly water soluble drugs, supersaturated drug delivery systems including solid dispersions and inorganic carriers have been suggested [1]. The aim of this study was to develop a small scale method to allow accurate determination of the release from complex carrier-mediated systems. For this purpose, a small scale method using the µDiss Profiler (pION INC, MA) to determine release rate in highly particle-rich suspensions, were developed. Fenofibrate was selected as model compound for this study. All measurements were performed at 37 °C, using a stirring rate of 100 rpm and 15 mL preheated fasted state simulated intestinal fluid (FaSSIF), following a previously published protocol [2]. Upon studies of high concentrations of the inorganic mesoporous carrier, Upsalite®, interference of the absorbance spectra was seen leading to inadequate release profiles when using the µDiss Profiler. To avoid the disturbance, nylon woven net filters (a kind gift from Merck Millipore, Darmstadt, Germany) for protection of the in situ UV-probes were evaluated. The release profiles of fenofibrate loaded Upsalite (FENO-Upsalite) were studied in various concentrations up to 1 mg/mL of FENO-Upsalite. A large interference of the measurement was seen for FENO-Upsalite and at a concentration of 1 mg/mL extensive shaking of the probes was needed to minimize the accumulation on the probe heads and reduce the interference. When using filters, no accumulation of particles was observed on the probe heads and hence, the interference was diminished. No significant adsorption was observed to the filters, as identified through measurements of comparative runs with crystalline FENO and FENO-Upsalite®. In conclusion, the µDiss Profiler is a promising method for the determination of release rate from complex and highly particle dense suspensions when the interference with spectra can be diminished. Reference [1] J. Brouwers, M.E. Brewster, P. Augustijns, J. Pharm. Sci. 98 (2009) 2549-2572. [2] S.B.E. Andersson, C. Alvebratt, J. Bevernage, D. Bonneau, C. da Costa Mathews, R. Dattani, K.

Edueng, Y. He, R. Holm, C. Madsen, T. Müller, U. Muenster, A. Müllertz, K. Ojala, T. Rades, P. Sieger, C. A.S. Bergström, J. Pharm Sci. (2016) DOI:10.1016/j.xphs.2016.03.010

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Impact of Polymorphism on the Development and Manufacturing of Pharmaceuticals

Rolf Hilfiker

Solvias AG, Kaiseraugst, Switzerland Tel +41 61 845 6000, Fax +41 61 845 6900, [email protected]

Solid-state properties of active substances and excipients are of major practical and commercial importance all the way from research to manufacture of the final product. The search for, and more importantly the selection of, the optimal solid form (salt, co-crystal, polymorph) is closely linked to the intended application of the final drug product. We will show what impact polymorphism can have commercially through intellectual property rights, what it can mean for patients due to manufacturing problems, and how it can delay registration by not meeting regulatory requirements. In connection with various solid forms, the first challenge is clearly identifying all relevant solid forms of a certain compound. Principles and strategies of form screening will be dis-cussed, but an example will be presented where even the best strategies failed to identify the stable form. The second challenge is to understand the relationships between the various forms. In order to be able to select the optimal form for further development, not only the thermodynamic stability of the various forms have to be elucidated as a function of temperature, but also the kinetics of conversion need to be studied. Using hydrate/anhydrate pairs as an example we will show how to determine phase diagrams of such systems as a function of tempera-ture and water activity and what the implications are in terms of form selection. The desired form then has to be produced in an efficient, safe and completely reliable process. In the majority of cases, a seeded crystallization process is the best way to achieve control over the obtained product. Examples will be presented and potential problems such as cross-seeding will be briefly discussed. Finally, the obtained solid product has to be thoroughly characterized. Characterization of solids is a particular challenge since, unlike liquids and gases, their properties are not isotropic. Furthermore, a solid can be inhomogeneous, so that the characteristics of any volume element may be different from another. If products from crystallization processes are considered, the product should be homogeneous on a medium scale, but it will still be necessary to determine both the intrinsic properties of the solid as well as the particle shape, particle size, and particle size distribution in addition to the mechanical properties of the solid such as powder flow properties. Accordingly, a vast number of characterization methods is available and required to fully understand the behavior of a solid. Further processing steps such as micronization may further change the solid, such as creating amorphous domains which may greatly impact the properties of the product. Quantification of such amorphous fractions is therefore important in order to be able to optimize the process in such a way that the amorphous fraction is minimized. In summary, the existence of various solid forms both represents a fantastic opportunity to create a product with optimized properties, but it also represents a big experimental and theoretical challenge, to find the relevant forms, characterize them, understand them and produce them.

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Cocrystal Solubility-pH Data Analysis using pDISOL-X (with Automatic Equilibrium Equation Generation)

Alex Avdeef

in-ADME Research, 1732 First Avenue, New York, USA; [email protected]

PURPOSE: Practically-insoluble active pharmaceutical ingredients (API) generally have poor oral bioavailability. In attempting to overcome this, a relatively new and still emerging formulation strategy is based on using pharmaceutical cocrystals (CC), which are crystalline solids, composed of the API and a water-soluble molecule, the ‘coformer’ (CF). The solubility of a CC is characterized by the solubility product, Ksp, with the product components in their uncharged forms, being associated in the crystal by complementary H-bonds. The purpose of this study was to develop a general CC solubility-pH data simulation/refinement procedure to rationalize the aqueous solution behavior of CCs as a function of pH, (a) with all required equilibrium equations auto-generated, (b) with auto-ionic-strength equilibrium constant compensation (activity coefficients calculated by the Stokes-Robinson (1948) hydration theory), and (c) with auto-ionic-strength compensation for the four Avdeef-Bucher (1978) pH electrode calibration parameters. The study augments the work of Naír Rodríguez-Hornedo et al. in their pioneering studies of the solubility-pH behavior of CCs. METHODS: A novel dual (API, CF) solubility-pH analysis computer program, pDISOL-XTM, has been developed. The unique mass-action algorithm does not require any explicitly derived solubility equations based on extensions of the Henderson-Hasselbalch equation. Rather, the program internally derives implicit equations, given a test set of equilibria and estimated constants (which are then iteratively refined by weighted nonlinear regression). The program was extensively tested, drawing on nearly all the published CC log S-pH data from the literature (largely from Rodríguez-Hornedo et al.), with equilibrium API and CF solubilities measured under the eutectic conditions (stable presence of CC and API solids in solution). RESULTS: The following cocrystal literature data analysis will be described in detail: carbamazepine-4-aminobenzoic acid (2:1), carbamazepine-saccharin (1:1, 2 studies), carbamazepine-salicylic acid (1:1), carbamazepine-cinnamic acid (1:1), indomethacin-saccharin (1:1), nevirapine-maleic acid (1:1), nevirapine-saccharin (2:1), nevirapine-salicylic acid (2:1), and gabapentin-3-hydroxybenzoic acid (1:1). The API thermodynamic solubility was elevated only slightly in most of the carbamazepine systems, with the formation of complexes between the API and charged forms of the CF. The carbamazepine-cinnamic acid CC showed a substantial elevation in the API solubility above pH 5. The nevirapine CCs did not indicate increases in API solubility. CONCLUSION: The advantages of CCs are more subtle than simply elevating thermos-dynamic solubility of the API. The dissolutions of CCs often lead to supersaturated solutions, which could increase oral bioavailability. CCs may form stable solids, with shelf life advantages over traditional formulations. The increased stability may improve the ta-bletability of the CC in the final formulation. The CC field is still in its infancy. It is expected that the data refinement and simulation tool, pDISOL-X, could help in understanding the solution dynamics of CC systems, and perhaps in the design of future systems.

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Two Novel Cocrystals with Improved Solubility and in Situ Monitoring of Cocrystallization of Lamotrigine

Shichao Du1,2,3*, Yan Wang1,2, Jingkang Wang1,2, Junbo Gong1,2,3**

E-mail: *[email protected]; **[email protected]

1National Engineering Research Center of Industry Crystallization Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China;

2The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin, Tianjin University, Tianjin, China;

3Key Laboratory Modern Drug Delivery and High Efficiency in Tianjin University, Tianjin, China

Lamotrigine is a widely used anticonvulsant drug with poor solubility and limited bioavailability. Crystal engineering strategy was applied to improve the physicochemical properties of lamotrigine in this work. Two novel cocrystals including lamotrigine 4,4’-dipridy cocrystal (2:1) and lamotrigine 2, 2’-dipridy cocrystal (1:1.5) were successfully screened by neat grinding and liquid assistant grinding. The novel cocrystals were fully characterized by powder X-ray diffraction (PXRD), differential scanning calorimeter (DSC), thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR) and Raman, specifically, the PXRD, DSC and TGA helped vertify the novel multicomponent crystals and determine their purity, and the FTIR and Raman helped confirm the novel multicomponent crystals belong to cocrystals. Then the novel cocrystals were also obtained by slurry crystallization. Process analytical technologies including focused beam reflectance measurement (FBRM), attenuated total reflectance fourier transform infrared (ATR-FTIR) and Raman were applied to investigate the cocrystallization process. The mechanism of cocrystal formation during solution-mediated phase transformation is discussed. Moreover, HPLC analysis showed that the dissolution rate and the solubility of the two novel cocrystals were both improved.

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Drugs as Materials: Processing, Structure, Properties

Elena Boldyreva

Institute of Solid State Chemistry and Mechanochemistry SB RAS, ul. Kutateladze 18, Novosibirsk, 630128, [email protected]

To become a drug, a pharmacologically active compound must be prepared in a specific form. It is in this form that the drug must be manufactured, packaged, stored, transported, administred and delivered to a target in the body. Whereas drugs move around the body in solution and act on the cellular and molecular level, the properties of their physical form need to be identified and optimized for in vivo performance, reliable manufacture and the protection of intellectual property. To successfully prepare a drug form that will be robust through manufacturing, stable before administration and then active with high bioavailability after administration, one needs to produce solid forms with a specific crystal structure and controlled particle size and shape, often as a multi-component composite. From a legal standpoint, it is important to note that in addition to active pharmaceutical ingredients (APIs), drug forms are also patentable. The development and control of drug forms is therefore crucial to many issues in intellectual property. Considering drugs as materials, one can apply the knowledge of solid-state chemistry and materials science and non-ambient conditions to obtain solid forms with optimized properties. These conditions include, among others, different types of mechanical and ultrasonic treatment, hydrostatic compression, high-temperature or cryogenic spray-drying, and crystallization from supercritical solvents. Solid-state reactions (e.g. dehydration or clathrate decomposition) can be efficient in accessing metastable polymorphs or in micronizing the sample uniformly. To achieve control over drug forms and the processes used for their robust manufacturing, one needs to take into account both the thermodynamic and kinetic aspects of their transformations. The aim of this lecture is to provide a guide through the vast ocean of publications, and to give a personal view of the problem mainly, but not exclusively, from the experience of my research group [1]. I acknowledge support from Russian Science Foundation, grant 14-13-00834. [1] E. Boldyreva, Current Pharm. Design (2016) in press.

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The Importance of Proton Transfer in Amorphous Active Pharmaceutical Ingredients

Marzena Rams-Baron, Żaneta Wojnarowska, Mateusz Dulski, Patryk Włodarczyk, Marian Paluch

Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland Silesian Center for Education and Interdisciplinary Research, 75 Pulku Piechoty 1a, 41-500

Chorzow, Poland

There are many active pharmaceutical ingredients that can exchange into various tautomeric forms when the long-range ordering, typical for crystal lattice is lost (e.g. in amorphous state). It is due to proton transfer reaction leading to the formation of drug with overall properties being different in comparison to initial product. The consequences of tautomerization interconversion cannot be ignored, however, due to changeable character and substantial sensitivity to external condition (temperature, pressure, solvent, pH) its detailed investigation can be challenging. Such difficulties may be overcome when we apply broadband dielectric spectroscopy (BDS), an approach commonly used in molecular dynamic studies. Since proton transfer leads to changes in viscosity or dipole moment of the sample, we can easily monitor the kinetics of reaction by BDS technique. In the past we detected proton transfer in several amorphous pharmaceuticals as well as biologically relevant materials. Mostly we observed that the ability to tautomerization can positively impact the stability of amorphous material. For example, proton transfer in glibenclamide drug resulted in higher population of amide form being characterised by better stability. Recently we have shown that in amorphous etoricoxib the existing tautomers can interact with each other via hydrogen bonds leading to heterodimer formation. Consequently, the crystallization tendency of such binary system was strongly reduced. Our latest results confirmed the presence of proton transfer in amorphous bicalutamide but contrary to previously studied materials the drug stability was limited and its recrystallization occured. So the effect of proton migration on amorphous drug stability depends solely on the material. To show the dual nature of tautomerization phenomenon and to explain its different impact on the stability of amorphous pharmaceuticals we will discuss the results of our recent studies concerning two mentioned APIs, etoricoxib and bicalutamide. The analysis of tautomerization kinetics together with results of theoretical and infrared spectroscopy studies will shed more light on this interesting phenomenon.

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Mechanism-Based Selection of Stabilization Strategy for Amorphous Formulation

Khadijah Edueng*,**, Denny Mahlin*, Christel Bergström*

*Department of Pharmacy, Uppsala University, Sweden **International Islamic University Malaysia

The aim with this study was to develop a method that can identify crystallization mechanism (solid-to-solid or solution-mediated) by which crystallization of amorphous compounds take place in solution. For this purpose, an experimental protocol based on differential scanning calorimetry (DSC), dynamic vapour sorption (DVS), polarized light microscope (PLM) and small-scale dissolution apparatus (pION µDiss) was established to investigate the mechanisms. This information was used to decide on how to stabilize amorphous formulations. Indapamide, metolazone, glibenclamide and glipizide were selected as model compounds and HPMC (Pharmacoat 606) and PVP (K30) were explored as the stabilizing polymers. From the analyses, amorphous indapamide, metolazone and glibenclamide were found to crystallize via solution-mediated nucleation while glipizide suffered from solid-to-solid crystallization. The addition of low amount HPMC (0.01 % and 0.001 % w/v) into the dissolution medium successfully prevented or delayed the nucleation of indapamide and metolazone during dissolution. On the contrary, low concentration of HPMC only reduced the crystal growth rate for glibenclamide. Amorphous solid dispersion of glipizide and PVP (K30) at a ratio of 50:50 % w/w was successful in minimizing solid-to-solid crystallization of glipizide, leading to an overall improvement in its dissolution rate and level of supersaturation. To conclude, a new experimental protocol was established that enables identification of crystallization mechanisms and provides a scientific rationale on stabilization of amorphous formulations.

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Discovery and Development of Pyrotinib: A Novel Irreversible EGFR/HER2 Dual Tyrosine Kinase Inhibitor with

Favorable Safety Profiles for the Treatment of Breast Cancer

Xin Li*, Changyong Yang**, Hong Wan*, Ge Zhang**, Jun Feng*, Lei Zhang*, Xiaoyan Chen***, Dafang Zhong***, Liguang Lou***, Weikang Tao*,

Lianshan Zhang**,****

*Shanghai Hengrui Pharmaceutical CO., LTD., 279 Wenjing Road, Shanghai 200245, China **Jiangsu Hengrui Medicine CO., LTD, Jiangsu, Lianyungang 222047, China

***China Pharmaceutical University, Jiangsu Key Laboratory of Drug Design and Optimization, Nanjing 210009, China

****State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

The discovery and development of a novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor SHR1258 (pyrotinib) for the treatment of HER2-postive breast cancer is presented. The structure-activity relationship of lead series and their pharmacokinetic properties were evaluated to identify the potential candidates for further in vivo efficacy studies and preclinical safety assessments. Metabolic pathway and drug-drug interaction were also investigated in preclinical settings. In particular, major metabolites in human and animal species were assessed with regard to potential toxicity or off-target side effects. Overall, the potent and selective EGFR/HER2 dual inhibitor, pyrotinib, displayed robust anti-tumor effects on HER2-overexpressing xenograft models and sufficiently safety windows in animals as well as favorable pharmacokinetic properties in human, which substantially ensures current clinical development. Finally, recent advances of pyrotinib in clinical studies are highlighted with very encouraging outcomes in patients with HER2-postive advanced breast cancer.

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Epiberberine, a Natural Protoberberine Alkaloid, Inhibit Urease of Helicobacter pylori and Jack Bean via Binding to the Sulfhydryl Groups

Lihua Tan, Cailan Li, Hanbin Chen, Zhizhun Mo, Jiangtao Zhou, Yuhong Liu, Zhilin Ma, Yuyao Xu, Xiaobo Yang*, Jianhui Xie*,, Ziren Su

School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, P. R. China

*Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, The Second Affiliated Hospital, Guangzhou University of Chinese

Medicine, Guangzhou 510120, PR China Corresponding authors: [email protected] & [email protected]

In our previous study, Rhizome Coptidis and its major component berberine were found to inhibit urease from Helicobacter pylori (HPU) and Jack bean (JBU). The anti-urease activity of Rhizome Coptidis was remarkable even superior to that of berberine. In continuation of our work, the present study was designed to further elucidate the urease inhibitory activities of five major protoberberine alkaloids in Rhizome Coptidis, namely berberine, palmatine, coptisine, epiberberine, jateorhizine. Results indicated that the five protoberberine alkaloids acted as concentration-dependent inactivators of urease with IC50 values ranging between 3-5087 μM for HPU and 2.3->10000 μM for JBU, respectively. Notably, epiberberine (EB) was found to be the most potent inhibitor against both ureases with IC50 values of 3.0 ± 0.0 μM and 2.3 ± 0.0 μM for HPU and JBU, respectively, which was more effective than the standard urease inhibitor, acetohydroxamic acid (41 ± 0.0 μM for HPU and 25 ± 0.0 μM for JBU, respectively). Further kinetic analysis revealed that the type of EB inhibition against HPU was slow-binding and uncompetitive, with Ki of 110 ± 0.006 μM, while slow-binding and noncompetitive against JBU with Ki of 4.6 ± 0.002 μM. Addition of thiol protectors, such as L-cysteine, glutathione and dithiothreitol, gradually abolished the inhibition, while Ni2+ competitive inhibitors, boric acid and fluoride, synergetically inhibited urease with EB, indicating the obligatory role of the active site sulfhydryl group for the inhibition, which was further supported by the molecular docking studies. In addition, binding of EB with the urease proved to be reversible, as about 65 % and 90 % enzymatic activity of HPU and JBU, respectively, could be restored by dithiothreitol application. These findings highlighted the potential role of Rhizoma Coptidis protoberberine alkaloids, especially EB, as a lead urease inhibitor in the treatment of diseases associated with ureolytic bacteria. Thus, EB had good potential for further development into a promising therapeutic approach for the treatment of urease-related diseases. Reference [1] C.L. Li, J.H. Xie, X.Y. Chen, Z.Z. Mo, W. Wu, Y.E. Liang, Z.Q. Su, Q. Li, Y.C. Li, Z.R. Su, X.B. Yang,

Planta Med. 82 (2015) 305-11.

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Wen Zhang, Xiaohui Hu, Shao-Lin Zhang, Kin Yip Tam

Drug Development Core, Faculty of Health Sciences, University of Macau, Macau, China Phenyl butyrate (PB) has been proved to decrease pyruvate dehydrogenase (PDH) phosphorylation level and increase PDH activity by inhibiting pyruvate dehydrogenase kinases (PDKs) in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibitor mode of action. As PDK1/PDH controls the critical switch between oxidative phosphorylation (OXPHOS) and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might depend on suppressing PDKs and promoting PDH activity. We showed that PB directly inhibited the kinase activity of PDK1 in a purified system. In cancer cells, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization and induced the apoptosis of cells. In summary, PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.

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A Potential Target for Parkinsonism Treatment

Tao Chen, Bo Jiang, Yuan Xu*, Feng Zhi*, Yiling Yang*, Ying Xia

Department of Neurosurgery, The University of Texas McGovern Medical School, Houston, Texas, USA

*Modern Medical Research Center, Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China

Parkinson’s disease (PD) is characterized by the insufficient production of dopamine in the substantia nigra of the midbrain. Although the etipathogenesis of PD is complex, the vulnerability of midbrain dopaminergic neurons to genetic defect, hypoxic/ischemic injury, and environmental neurotoxins affecting the dopamine biosynthetic pathways has long been implicated as a potential cause of PD. Our serial studies have shown that the δ-opioid receptor (DOR) is uniquely expressed in the cortex and striatum in high density and plays an important role in protection against neuronal injury. More recently, we found a major decrease in DOR expression with a significant increase in α-synuclein in the striatum of the transgenic mice expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) that exhibit late-onset and progressive hypokinetic motor deficits with age-dependent loss of dopaminergic neuron in substantia nigra. Furthermore, in an in vitro cell model, we found that MPP+ treatment led to a marked reduction in DOR expression with α-synuclein overproduction/aggregation and cell injury. DOR activation with UFP-512, a potent and specific DOR agonist, greatly reduced α-synuclein overproduction and aggregation and attenuated cell injury, while DOR antagonism reversed the protective effect. Furthermore, we found that in the in vitro model, hypoxia and MPP+ reduced cell viability, progressively suppressed the expression of PINK1 protein and increased the cleaved caspase 3. DOR activation with UFP-512 effectively protected the cells from hypoxia and/or MPP+ induced injury, reversed the reduction in PINK1 protein and significantly attenuated the increase in the cleaved caspase 3 that plays a central role in the execution-phase of cell injury. Our data suggest that DOR may function as a protector against α-synuclein overproduction/aggregation and caspase 3 insult and increasing DOR activity is likely an effective strategy for the prevention and treatment of parkinsonism conditions.

This work was supported by Memorial Hermann’s Foundation, Vivian L Smith Neurologic Foundation, NIH (AT-004422), the Natural Science Foundation of China (81560202), the Natural Science Foundation of Hainan Province (2016CXTD010) of China and Jiangsu SPMS (BL2014035) of China.

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Excessive Porphyrin Production Induced by Cyclohexylmethyl Flavonoids Suppresses Self-Renewal Propagation of Breast Cancer Stem Cells

Wen-Ying Liao1,2, Chih-Chuang Liaw4, Chien-Shu Chen3, Sheng-Chu Kuo2,3, Yuan-Chao Huang2, Chia-Ning Shen2,5

1Genomics Research Center, Academia Sinica, Taipei 115, Taiwan 2Graduate Institute of Pharmaceutical Chemistry

3School of Pharmacy, China Medical University, Taichung 402, Taiwan 4Department of Marine Biotechnology and Resources,

National Sun Yat-sen University, Kaohsiung 804, Taiwan 5Graduate Institute of Clinical Medicine, Taipei Medical University,

Sinyi District, Taipei 110, Taiwan

Our recent studies have shown that porphyrin homeostasis maintained by ABCG2 tranporter regulates self-renewal of embryonic stem cells and epidermal stem cells of adult skin[1,2]. However, the consequence of disrupting porphyrin homeostasis in cancer stem cells is less clear. Breast cancer stem cells express functional ABCG2 transporters that mediating multidrug resistan and display a panel of stem cell marker including CD44 and SSEA3[3]. We initially found that the ABCG2-expressing subpopulation of MCF-7 cells is able to efflux protoporphyrin IX (PPIX). Functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle. Based on development of porphyrin-efflux assay, we screened a group of natural flavonoids[4,5] and identified several cyclohexylmethyl flavonoids that can effeciciently inhibit ABCG2 activity possibly via binding to its nucleotide-binding domain and inhibiting its ATP hydrolysis activity. Among these flavonoids, ugonins J and K with a non-polar cyclohexylmethyl group were found to be able to efficiently suppress self-renewal propagation of ABCG2-expressing CD24-

/lowCD44+ cell subpopulation. This suppression was also due to disruption of porphyrin homeostasis, which led to elevated levels of reactive oxygen species which in turn activated p53 pathway. These results suggest that the porphyrin-efflux assay could be used as a method for searching lead compounds to target to cancer stem cells, and also that flavonoids with a non-polar bulky group can be useful in cancer therapy by suppressing the propagation of breast cancer stem cells via disruption of porphyrin homeostasis.

References [1] J. Susanto, Y.H. Lin, Y.N. Chen, C.R. Shen, Y.T. Yan, S.T. Tsai, C.H. Chen, C.N. Shen, PLoS One 3

(2008) e4023. [2] H. M. Chang, W. Y. Huang, S. J. Lin, W. C. Huang, C. R. Shen, W. Y. Mao, C. N. Shen, Exp.Dermatol.

2016 Jan 6. doi: 10.1111/exd.12936. [Epub ahead of print] [3] S. K. Cheung, P. K. Chuang, H. W. Huang, W. W. Hwang-Verslues, C. H. Cho, W. B. Yang, C. N. Shen,

M. Hsiao, T. L. Hsu, C. F. Chang, C. H. Wong. Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 960-965. [4] Y. C. Huang, T. L. Hwang, C. S. Chang, Y. L. Yang, C. N. Shen, W. Y. Liao, S. C. Chen, C. C. Liaw. J.

Nat. Prod. 72 (2009) 1273-1278. [5] W. Y. Liao, C. C. Liaw, Y. C. Huang, H. Y. Han, H. W. Hsu, S. M. Hwang, S. C. Kuo, C. N. Shen, Evid.

Based Complement. Alternat. Med. 2013 (2013) 170261.

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http://www.ncbi.nlm.nih.gov/pubmed/19107196


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Predictors for Drug Effects with Alzheimer’s Disease: Shed New Light from EEG Parameters to Brain Connectomics

Li Yang, Zhongyan Wang, Huiling Zhang, Wei Xu, Shuxing Zheng, Haiyong Zhang, Yin Tian*

College of Bio-information, ChongQing University of Posts and Telecommunications, ChongQing, 400065, China; *[email protected]

Alzheimer's disease (AD), a form of dementia, is a chronic neurodegenerative disease affecting the patient's life and social skills greatly. Patients suffer from memory and cognitive impairments and there is no cure for this disease. As AD has become more common in the world, researchers spent a lot of effort to develop treatments for AD. A large number of studies have put forward the pathogenesis of AD such as cholinergic deficit hypothesis, amyloid cascade hypothesis, tau protein abnormalities and inflammation. Among these paradigams, the amyloid-β (Aβ) peptides aggregation and tau hyperphosphorylation are thought to play key roles in AD. Simultaneously, pharmaceutical industry is challenged by low clinical success rates for new AD drug registration. The reasons why pharmaceutical industry fails to develop effective drug treatment for AD are as follows: (1) the underlying mechanisms remain unclear. (2) The poor translation of drug efficacy data from animals to humans, including the validity of animal models, toxicological issues, and pharmacodynamics (PD) and pharmacokinetics (PK). (3) Treatment outcomes lack robust PK endpoints. (4) Good brain exposure is not always achievable due to the blood brain barrier (BBB). (5) Hypotheses are considered irrelevant. Nevertheless, before a new drug developed, the investigative compounds need to undergo a series of PD and PK tests. Thus, biomarkers are required to act as surrogate end points and predictors of drug effects in order to reflect the progression of AD. Multimodal imaging technology including magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used in the clinics. Electroencephalogram (EEG), generating from the result of the postsynaptic potential discharge between cells, could be a potential measure to bridge the gaps between animal and human data. Brain connectomics is a kind of biotechnology that fully and finely describe human brain network from micro to macro, structure to function and detect the pattern of the network connection. Here we discuss recent evidence on EEG characteristics (i.e. how EEG works in rodents and humans, what parameters to check out, and how they are relate to PD) and brain connectomics (i.e. small-world network, shortest path length, clustering coefficient, glob effect, and local effect) as core biomarkers in combined use with preoperative clinical data. Compared to traditional biomarkers, EEG is a real-timing detection technique to rationalize the brain connectomics, which based on brain network for monitoring of the drug effects on animal models and humans. To emphasize the contribution of EEG and brain connectomics to preclinical/clinical studies, we cited some recent studies in which EEG and brain connectomics are applied to monitor the drug effect and measure cognitive changes. It is expected that this novel approach will offer a deeper understanding on the drug efficacy at a microcirculatory level, which will be useful to support the development of new AD treatments and AD.

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Data Mining for Equilibrium Solubility of Ionizable Druglike Molecules

Alex Avdeef

in-ADME Research, 1732 First Avenue, New York, USA; [email protected]

PURPOSE For typical druglike molecules, it had been often assumed that the interlaboratory reproducibility in measured solubility is about 0.6 to 0.7 log unit. It will be shown that much better results can be produced in data mining for training sets used in the in silico prediction of solubility. Two ways of improving the usefulness of published solubility-pH measurements will be described, consisting of normalizing the data for ionization and temperature effects. Also, ways to improve the quality of future measurement of equilibrium solubility will be briefly discussed. METHODS The “gold standard” shake-flask (SF) and related methods used to measure equilibrium solubility of ionizable drug-like compounds as a function of pH were examined, by studying hundreds of publications (848 references). The solubility-pH analysis computer program, pDISOL-XTM, has been extended to calculate (a) intrinsic solubility (S0) and saturation pH (pHsat) from solubility measurements done in pure water with unspecified pH, and (b) to transform S0 values determined at various temperatures to that at a single reference temperature (25 or 37 oC). RESULTS Useful methods to improve the quality of mined solubility data have been identified. A few examples of interlaboratory/intertemperature log S0

25 (molarity scale) values include acetylsalicylic acid: -1.69 ± 0.09 (n=13); ampicillin: -1.62 ± 0.13 (n=10); barbital (3 polymorphs): -1.42 ± 0.04 (n=11); caffeine: -0.96 ± 0.08 (n=10); ciprofloxacin: -3.64 ± 0.15 (n=11); cyclosporin A: -5.00 ± 0.14 (n=5); dexamethasone: -3.65 ± 0.08 (n=4); diclofenac: -5.36 ± 0.19 (n=26); ethisterone: -5.66 ± 0.06 (n=4). CONCLUSION With the aid of the new data analysis algorithm and improved methodology, it becomes evident that the currently expected poor interlaboratory reproducibility can be improved to 0.06 to 0.17 log unit, with higher errors for the least-soluble druglike molecules. Such improvements in data mining are expected to contribute to more reliable in silico prediction models of solubility for use in drug discovery.

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How to Optimize the Physicochemical and Biomimetic Properties of Putative Drug Molecules?

Klara Valko

Bio-Mimetic Chromatography, Unit 5H Business & Technology Centre, Stevenage, Herts SG1 2DX, UK

Potent putative drug molecules often fail at later stages of the drug discovery process due to poor absorption, distribution and pharmacokinetic properties. Physicochemical properties of molecules are responsible for the distribution and nonspecific binding of the molecules in vivo. The in vivo experiments are resource intensive and can be carried out only for a small number of compounds. Alternatively, biomimetic chromatographic measurements provide a platform for easy characterization of large number of molecules by using components from the body such as proteins and phospholipids1. Due to dynamic distribution processes during chromatography the compounds have different retention times on chemically bonded protein and phospholipids (Immobilized Artificial Membrane, IAM) stationary phases. The retention behaviour of compounds can be easily studied under various conditions (for example different pHs) thus revealing the acid/base character of the compound. The structure – biomimetic retention studies revealed that compounds bearing positive or negative charges bind differently to albumin and phospholipids causing different volume of distribution and tissue plasma partition that octanol/water lipophilicity are not able to model well. From the systematically measured retention data we can estimate the in vivo distribution of the compounds, such as volume of distribution, tissue/plasma partition, drug efficiency and the free concentration relative to dose that is available at the site of action. When the potency of the compound is known we can carry out early dose estimation without any animal experiment. These mechanistic mathematical models have been published2 and validated using the clinical data of known drug molecules and they have been utilized over 30 drug discovery programs at GSK. References: 1. K. Valkó, J. Chromatogr. A. 1037 (2004) 299–310. doi:10.1016/j.chroma.2003.10.084. 2. K. Valkó, Physicochemical and Biomimetic Properties in Drug Discovery: Chromatographic

Techniques for Lead Optimization, Wiley Hoboken NJ, 2014.

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Comparison and Modelling of Experimental and Calculated Distribution Coefficient (Logd) Values at Various pHs

Mare Oja, Uko Maran

Institute of Chemistry, University of Tartu, Estonia Partition coefficient (logP) is widely used parameter in drug discovery and toxicology to describe compounds permeability properties. LogP describes partition of neutral compounds, although in real environment most of compounds are partially charged. This makes important to take account of ionization and pH, which can be achieved by using distribution coefficient (logD) at certain pH. LogD is usually calculated using logP and dissociation coefficient, but this presumes that these values are correct to guarantee the accuracy of logD values. Unfortunately, literature provides experimental logD values only for pH 7.4 and almost no data is available for other pH-s, which prohibits the use of logD as parameter for permeability. This presentation gives overview of available logD calculators and new prediction models for logD of various pH-s. For this logD values for pH 3, 5, 7.4 and 9 have been measured for ~200 compounds using miniaturized shake-flask method. Obtained original experimental data has been compared with predicted values from different predictors. These relationships are analysed based on chemical classes. Also measured experimental logD values are used to develop new quantitative structure-activity relationships (QSAR) for selected pH-s. New proposed models and their descriptor content have been analysed. Acknowledgement: Estonian Ministry for Education and Research (grant IUT34-14)

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Quantitative Structure Activity Relationship (QSAR) Analysis of Indirubin Derivatives as Cyclin Dependent Kinase 5 (CDK5) Inhibitor

Muhammad Arba, Rahmad Sutrisna, Ruslin

Faculty of Pharmacy, Universitas Halu Oleo, Kendari-Southeast Sulawesi, Indonesia, 93231 Email: [email protected]

In order to gain further insights into the structural requirements for CDK5 inhibitor by indirubin derivatives, two dimensional QSAR on a data set consisting of structurally diverse compounds reported as CDK5 inhibitor was performed. Structural optimization was conducted using semi-empirical AM1 method, while calculation of 13 descriptors representing the hydrophobic, electronic, and steric parameters was achieved using MOE2009.10. The developed QSAR model selected has correlation coefficient r2 = 0.788, r = 0.887, and cross-validated squared correlation coefficient q2=0.56. Using the model, we designed 25 indirubin derivatives and 18 of which showed better prediction activity. Reference 1. J. Begum, V. T. Skamnaki, C. Moffatt, N. Bischler, J. Sarrou, A-L. Skaltsounis, D. D. Leonidas, N. G.

Oikonomakos, J. M. Hayes, J. Mol. Graph. Model. 61 (2015) 231–242.

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Design and Molecular Docking Study of Novel Indirubin Derivatives As Inhibitor of Cyclin Dependent Kinase 5 (CDK5)

Ruslin, Rahmad Sutrisna, Muhammad Arba

Faculty of Pharmacy, Universitas Halu Oleo, Kendari-Southeast Sulawesi, Indonesia-93231 [email protected]

The cyclin-dependent kinase 5 (CDK5) is a serine/threonine protein kinase playing a pivotal role in the the development of neurodegenerative diseases such as Alzheimer’s disease. In the present work, we have designed a series of indirubin derivatives as CDK5 inhibitor. Molecular docking studies were also performed wherein a total 18 indirubin derivatives were docked into the active site of the CDK5 crystal structure to analyze ligand-receptor interactions. Docking analysis yielded 8 best ligands which have better binding affinity than the parent compund. Interaction analysis indicated that each best ligands were accomodated in the active pocket of CDK5 and share the same key interaction as native ligand. The result showed that some designed ligands could be very useful in the development of CDK5 inhibitor. Reference 1. M. Mapelli, L. Massimiliano, C. Crovace, M.A. Seeliger, L.-H. Tsai, L. Meijer, A. Musacchio, J. Med.

Chem. 48 (2005) 671-679.

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Strategies for the Development of Liquid, Semisolid and Solid Lipid-Based Drug

Delivery Systems

Abu T. M. Serajuddin

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens, NY 11439, USA

In recent years, there has been a great interest in the pharmaceutical field on lipid-based drug delivery systems for the oral bioavailability enhancement of poorly water-soluble drugs. They are usually developed as solutions with drugs dissolved in mixtures of lipids, surfactants and/or co-surfactants that form microemulsion with particle size <250 nm upon dilution with aqueous media of the gastrointestinal tract. The formulations are often referred to as microemulsion preconcentrates or self-microemulsifying drugs delivery systems (SMEDDS). In this presentation, a systematic approach of selecting lipid-surfactant mixtures (preconcentrates) that would spontaneously form microemulsions upon dilution with water will be discussed. Psuedoternary lipid-surfactant-water phase diagrams using mixtures of monoglyceride with either diglyceride or triglyceride will be presented to illustrate potential advantage of using mixed lipids. Certain combinations of lipids and surfactants produce gels once they come in contact with aqueous media and thus impede the formation of microemulsion. Strategies to overcome the gel formation will be presented. One major disadvantage with the development of self-emulsifying drug delivery system is that the preconcentrates used as formulations are generally liquids and have to be encapsulated in soft gelatin capsules (for example, Neoral, a Novartis product). Various strategies to convert microemulsion preconcentrates into solids, yet maintaining all attributes of liquids, will be discussed. The solidified formulation can be filled into hard gelatin capsules. In recent years, there has also been much interest on the conversion of liquid lipid-based formulations into tablets. However, there are only limited studies on the development of tablet dosage forms for lipid-based drug delivery systems reported in the literature. The poor tabletability of powders loaded with liquid lipids and surfactants has been the major issue in the development of tablet formulations. The results of our recent work on the development of tablet formulations of lipid-based systems will also be presented. We have identified that, among many silicates studied, Neusilin® US2 is the only silicate that is able to produce tablets with acceptable tensile strength in presence of the lipid component as high as 1:1 w/w ratio. Liquid is mostly adsorbed into the pores of the silicate rather than at the surface enabling compression into tablets. We also addressed the issue of incomplete drug release from various silicate formulations that had been reported in the literature. Strategies to ensure complete drug release from the silicate formulation will also be presented. Finally, how microemulsion formation may reduce cytotoxic effects of lipids and surfactants will be briefly discussed.

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Study on Effective Methods for Stabilizing the Amorphous Forms of Anti-Cholesterol Drug: Ezetimibe

Marian Paluch, Justyna Knapik

Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland

Ezetimibe is important drug inhibiting the intestinal absorption of both dietary and biliary cholesterol by impeding its passage across the intestinal wall. However, this drug exhibits very low bioavailability (35 %). The conversion of the crystalline to the amorphous state is one of the very promising way of enhancement of bioavaiability. Unfortunately this strategy suffer from one fundamental drawback – amorphous materials are physicaly unstable and they may simply revert to their more stable crystalline state. In this presentation, we will focus on the problem how to improve the physical stability of amorphous Ezetimibe. As a consequence three different methods leading to suppression of re-crystallization of amorphous ezetimibe will be presented [1-3]. Moreover we will provide explanation of possible molecular mechanism for this stabilization effect. References: 1. J. Knapik, Z. Wojnarowska, K. Grzybowska, L. Hawelek, W. Sawicki, K. Wlodarski, J. Markowski,

M. Paluch, Mol. Pharmaceutics 11 (2014).4280−4290 2. J. Knapik, Z. Wojnarowska, K. Grzybowska, K. Jurkiewicz, A. Stankiewicz, L. Tajber, M. Paluch,

Mol. Pharmaceutics 12 (2015).3610–3619 3. J. Knapik, Z. Wojnarowska, K. Grzybowska, K. Jurkiewicz, A. Stankiewicz, M. Paluch, Mol.

Pharmaceutics 13 (2016) 1308–1316.

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Theoretical Investigation on Bioequivalence and Biowaiver Scheme

Kiyohiko (Kiyo) Sugano

Toho University, Japan

Bioequivalence (BE) between a generic drug product and a brand-name drug product is critically important to guarantee the safety and efficacy of the generic drug product. Therefore, a rigid and valid rationale is required as the basis for the waiver of clinical bioequivalence studies (biowaiver). The oral absorption of a drug is determined by the dissolution rate, the does-to-solubilization capacity ratio (the dose number, Do), and the intestinal permeability of the drug. The elimination half-life (T1/2) also affects Cmax. In the literature, the biowaiver schemes have been discussed based on the biopharmaceutics classification system (BCS based biowaiver scheme (BCS-BWS)). However, the rationales behind BCS-BWS have been ambiguous, especially for the criteria for a dissolution test, due to the absence of the dissolution rate and T1/2 in BCS. The aim of this study was to provide theoretically rigid rationales for a biowaiver scheme. Two mutually complemental approaches, that is, analytical solution and computational numerical integration, were employed to investigate the dissolution test criteria (the critical dissolution number, Dncrit). Dncrit was defined as a value to show BE of AUC and Cmax against infinitely rapid dissolution (Dn = ∞). An analytical solution for Dncrit of Cmax BE was derived from the Fa equation by taking T1/2 into account. Numerical integration was used to calculate Dncrit accurately. Dncrit for Cmax was found to be dependent on T1/2 as well as Pn and Do (Figure 1). Dncrit of a BCS class I drug with a short T1/2 is larger than that of a long T1/2 drug. This point should be taken into account for a regulartory biowaiver guideline.

Figure 1. Dncrit caluculated. The gastrointestinal tract was represented by an one compartment

model. The dissolution of a drug was expressed by the Noyes-Whitney equation. The permeation of a drug was expressed by the first order equation. The BCS quadrant is shown on the undersurface.

.

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Anti-Tumour Effects of a Human & Mouse Cross-Reactive’ Anti-TACE Antibody in a Pancreatic Cancer Model In Vivo

Shun Ming Yuen*, Jie Ye*, Gillian Murphy*, Ruiyu Xie*, Hang Fai Kwok*,**

*Faculty of Health Sciences, University of Macau, Avenida de Universidade, Macau SAR **CRUK Cambridge Insititue, University of Cambridge, Cambridge CB2 0RE, U.K.

TNF-Alpha Converting Enzyme (TACE) is a membrane-bound zinc metalloprotease (MP) that may play a significant role in tumour biology, notably by the conversion of many inactive cell-surface ligands into active soluble ligands; e.g. ErbB-ligands such as TGF-Alpha, HB-EGF, EGF, Amphiregulin and Epigen. Moreover, TACE can stimulate local inflammation by solubilising TNF-Alpha, and aid immunological evasion by removing tumour cell surface MICA. Due to the homology between MP active sites, the development of small molecule TACE inhibitors has been plagued with unwanted non-specific MP activity. Recently, our group has successfully developed an inhibitory TACE antibody [A9(B8)] and showed the effective inhibition of both human and mouse TACE. In this study, we evaluated anti-tumour efficacy of A9(B8) -- the first specific ‘Human and Mouse Cross-Reactive’ anti-ADAM17 inhibitory IgG antibody with a mouse model of pancreatic ductal adenocarcinoma (PDA). It has been demonstrated that ADAM17 is required for Kras-deriven tumorigenesis in PDA mouse models. Here, we show that intraperitoneal injection of A9(B8) effectively attenuates high grade pancreatic intraepithelial neoplasisa (PanIN) formation in Pdx1Cre; KrasG12D; Trp53fl/+ mice. Thus, A9(B8) has anti-ADAM17 activity in vivo, inhibits pancreatic tumorigenesis and has potential for the use in ADAM17-dependent tumours.

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Design, Synthesis and Antitumor Activity of IMB-1506 Derivatives

Jun Zhang, Xiaoning Li

Institute of Medicinal Biotechnology, Nanwei Road No.2, Beijing, 10005

A series of novel IMB-1506 [1]derivatives were designed, synthesized[2] and evaluated for their in vitro antitumor activity against six human cancer cell lines by SRB assay. Results reveal that four target compounds have considerable antitumor activity against all the cell lines, which is more active than IMB-1506 and Sunitinib. Among them, compounds M3 (3.103 μM) and M6 (3.721 μM) were found to be about 10-fold more potent than the two references against A549 and SKOV-3, respectively.

References [1] Minghua Wang, Cheng Ye, Mingliang Liu. et al., Bioorg. Med. Chem. Lett. 25 (2015) 2782-2787. [2] Kai Lv, Li-Li Wang, Ming-Liang Liu et al., Bioorg. Med. Chem. Lett. 21 (2011) 3062-3065.

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Design, Synthesis and In vitro Antitumor Activity of Vandetanib Derivatives

Xiaoning Li, Jun Zhang

Institute of Medicinal Biotechnology, Nanwei Road No.2, Beijing, 100050 To design and synthesize a series of novel vandetanib derivatives, and evaluate their in vitro antitumor activity. Title compounds were synthesized through the condensation of 4-((4-bromo-2-fluorophenyl)amino)-6- methoxyquinazolines[1], which were prepared from 7-(benzyloxy)-6-methoxyquinazolin-4-ol, and piperidines obtained from N-Boc-3/4-piperidones[2]. Title compounds were evaluated for their in vitro antitumor activity by SRB assay. Twenty novel compounds were synthesized in this work. Among of them, seven compounds (at 30 μmol/L) were found to have > 65 % inhibitions against A549. Compounds 15b1 (IC50: 1.94 μmol/L) and 16b2 (IC50: 0.26 μmol/L) against A549 and compound 15b2 (IC50: 4.83 μmol/L) against KB were more active than or comparable to vandetanib. The structure skeletons at C-7 position of vandetanib were enriched. Compound 16b2 with 6 fold more activity than vandetanib against A549, is extremely worthy of further research. References [1] K. Lv, L. L. Wang, M. L. Liu et al., Med. Chem. Res. 22(4) (2013) 1723-1729. [2] P. A. Plé, T. P. Green, L. F. Hennequin et al. J. Med. Chem. 47(4) (2004) 871-887.

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Geniposide Improves Function and Reduces Apoptosis in High Glucose-induced Glucotoxic Insulinoma Cells

Lixia Guo*, Xuxu Zheng*, Jianhui Liu*,**, Zhongyi Yin*

*Chongqing Key Lab of Natural Medicine Research, Chongqing Technology and Business University, Chongqing 400067, China

**College of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400050, China.

Chronic exposure to hyperglycemia can lead to impaired glucose-stimulated insulin secretion (GSIS) and restrained proliferation and neogenesis in insulin producing β cells [1]. Our previous work showed that in pancreatic β cell line, geniposide decrease ATP production to inhibit GSIS induced by acute stimulation of a high concentration of glucose [2]. But the effect of geniposide on the impairment in function and mass of β-cells exposed to elevated levels of glucose is unknown. And the mechanism of action is not well understood. In the present study, it was tested how impaired GSIS and restrained proliferation observed in insulinoma INS-1 cells after prolonged culture at 33 mM glucose were affected by the inclusion of 1 or 10 μM geniposide during culture. Our results indicate that glucose-induced impairment of insulin release was significantly reverted by the inclusion of 1 or 10 μM geniposide. Furthermore, inclusion of 1 or 10 μM geniposide during culture induced phosphorylation of AMP-activated protein kinase (AMPK), which promotes utilization of nutrient stores for energy production. AMPK phosphorylation was paralleled by increased number of cells, increased expression of AMPK downstream target heme oxygenase 1 (HO-1), all of which were increased by high glucose concentration. In addition, geniposide protected rat insulinoma cells from apoptosis under high-glucose conditions. And these effects were associated with increased apoptosis-related proteins Bcl-2/BAX ratio. In conclusion, geniposide dose dependently improves β-cell function and increases proliferation in β cells exposed to prolonged hyperglycemia. Acknowledgements: This work was supported by Chongqing Science & Technology committee (CSTC, 2014jcyjA10048), the Natural Science Startup Foundation of Chongqing Technology and Business University (1352009) and the open project of Key Laboratory of Natural Medicine Research of Chongqing Education Commission (1456032, CQCM-2015-08). Reference [1] H.K. Nyblom, K. Thorn, M. Ahmed, P. Bergsten, Proteomics 6 (2006) 5193-5198. [2] J.H. Liu, L.X. Guo, F. Yin, Y.L. Zhang, Z.X. Liu, Y.W Wang, PLoS One 8 (2013) e78315.

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DSC as an Identification Tool for Protein Drugs and Protein Test Systems

Jamlet Monaselidze, Mikheil Gadabadze*, Maya Gorgoshidze, Maya Kiladze, David Khachidze, Shota Barbakadze

E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Georgia *Institute of Hematology and Transfusiology, Tbilisi, Georgia

On examples of (i) 20 % transfusion albumin (Octapharma Pharmazeutika, Produktionsges, mbH, Austria), (ii) Octagam 5 % sterilized solution mage from human plasma with total protein content of immunoglobulin G 95 %, including IgG1 60 %, IgG2 32 %, IgG3 7 %, and IgG4 1 %, (iii) EU Rituximab (Mabthera) that contains monocline IgG1, (iv) and Russian Acelbiya (Biocad) have been studied with Differential Scanning Microcalorimetry (DSC) in temperature rage 20°-105° C. It has been demonstrated that IgG1, IgG2, IgG3, and IgG4 melt at about 55°, 62°, 74°, and 81° C, accordingly, and their contents calculated according to DSC data coincide with manufacturers’ data within 7 % preciseness, taking into account that melting enthalpy of IgG is about 5 cal per gram protein. Also, we have demonstrated that the Rituximab (Mabthera) contains fewer impurities than Acelbiya (Biocad). We have calculated thermodynamic parameters of deconvoluted peaks of each above mentioned samples.

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Assessment of the Impact of Distribution and Storage Condition on the Quality of Medicines Marketed by National Medical Supplies Fund in

Sudan Healthcare Network

Elrashid Saleh Mahdi, Nawal Elhadi, Noon Jar Elnabi*, Gamal Khalaf Allah Mohd Ali

National Medical Supplies Fund, Federal Ministry of Health Khartoum 11111, Sudan *National Drug Quality Control Laboratory, Federal Ministry of Health, Khartoum, Sudan

Post market quality surveillance of selected products distributed by National Medical Supplies Fund was carried at central and regional levels in Sudan Healthcare Network. The objective of this study is to monitor the quality of products distributed by National Medical Supplies Fund. The criterion of selection of the medicines was risk assessment based. Specifically generic photosensitive, narrow therapeutic index products medicines and items imported from countries of week regulatory authorities. Potential seven items were identified and 22 samples were collected from various regional stores. The numbers of units collected as per sample were based on as determined by National Drug Quality Control Laboratory. A sample checklist was provided to sampling team prior to their departure to collection sites. Each collected sample was secured in a sealable plastic bag and attached to its corresponding sample collection form. The sample collection form contained all traceable data that accompanied the sample from the site of collection to National Medical Supplies Fund Headquarter. The samples were visually inspected and sent to the National Drug Quality Control Laboratory for confirmatory testing. The results of visual inspection and laboratory testing showed that the samples were complied with pharmacopeia of manufacturing. The study was concluded to products selected were quite stable in the distribution and storage conditions provided by National Medical Supplies Fund and distribution and storage conditions was satisfied the requirement of the selected products.

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The Ultrafine Formulation of Betulin with Biocompatible Carriers Exhibiting Improved Dynamics of Dissolution

Svetlana A. Myz*, Andrey G. Ogienko**, Anna A. Ogienko***, Tatyana P. Shakhtshneider*, Svetlana A. Kuznetsova****

*Institute of Solid State Chemistry and Mechanochemistry SB RAS, Novosibirsk, Russia; REC Molecular Design and Ecologically Safe Technologies, Novosibirsk State University

**Nikolaev Institute of Inorganic Chemistry SB RAS, Novosibirsk, Russia; Mendeleev College of Chemistry and Technology, Novosibirsk, Russia

***Institute of Geology and Mineralogy SB RAS, Novosibirsk, Russia ****Institute of Chemistry and Chemical Technology SB RAS, Krasnoyarsk, Russia; Siberian

Federal University, Krasnoyarsk, Russia

Betulin (lup-20(29)-ene-3β,28-diol, C30H50O2), pentacyclic triterpene alcohol produced from birch bark [1], is widely studied in recent years due to its biological activity such as anticancer, antiviral, antibacterial and hepatoprotective [2]. Because of its poor aqueous solubility, betulin has low and variable oral bioavailability. To increase both the dissolution rate and solubility of betulin, a variety of methods has been suggested. In this work, cryogenic technologies were used to develop a method for the preparation of ultrafine betulin formulations with the aim of enhancing the dissolution rates of betulin and related drugs that exhibit poor solubility in water. The formulation of betulin with PEG and β-glycine was obtained by freeze-drying. The prepared formulation was a light, fluffy powder, demonstrating very low bulk density. According to SEM data, the prepared formulation consists of two types of particles: perforated flakes with linear dimensions of 10-100 µm and thickness of approximately 100 nm, and small (1-5 µm) flat particles. The rate of dissolution of betulin from the formulation into water was significantly higher in comparison with the initial betulin sample as well as its composite with polyethylene glycol obtained by ball-milling. Acknowledgments: The work was supported by the Council for Grants of the President of RF for Support of Leading Scientific Schools (project no. NSh-8390.2016.3). References [1] S. Alakurtti, T. Mäkelä, S. Koskimies, J. Yli-Kauhaluoma, European Journal of Pharmaceutical

Sciences, 29 (2006) 1-13. [2] T. G. Tolstikova, I. V. Sorokina, G. A. Tolstikov, A. G. Tolstikov, O. B. Flekhter, Russian Journal of

Bioorganic Chemistry, 32 (2006) 37-49.

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The Approaches to Preparation of Betulin Esters Composites with Improved Biological Activity

Tatyana P. Shakhtshneider, Yuriy N. Malyar*, Mikhail A. Mikhailenko, Anna S. Zamay*, Natalia A. Pankrushina**, Svetlana A. Kuznetsova*

Institute of Solid State Chemistry and Mechanochemistry SB RAS, Novosibirsk, Russia; Novosibirsk State University, Novosibirsk, Russia

*Institute of Chemistry and Chemical Technology SB RAS, Krasnoyarsk, Russia **Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk, Russia

Betulin esters, betulin diacetate (BDA) and betulin dipropionate (BDP), were obtained directly from the outer bark of birch bark according to the original methods[1,2]. It is known that they posess a versatile biological activity. The poor solubility of betulin esters in water has limited their applications. The purpose of this work was to obtain the composites of betulin esters with improved dissolution properties and study their pharmacological activity. The composites of BDA and BDP with water-soluble biopolymer arabinogalactan (AG) isolated from larch wood [3] were prepared by ball-milling the mixtures of the components in a SPEX 8000 mixer mill (CertiPrep Corp., USA). In the case of the mechanocomposites, the solubility of betulin esters increased due to disordering of crystal structures of the drugs and formation of molecular complexes with the polymer. The composites obtained exhibited antitumor properties against the ascites adenocarcinoma Ehrlich cells. The composites of betulin esters with AG were prepared also as amorphous water-soluble films by dissolution the mixtures of the components in water and subsequent solvent evaporation. The composites prepared as the the films exhibited the highest antitumor activity against lung adenocarcinoma in comparison with the initial substances[4]. The microwave (MW) irradiation was successfully used to obtain a supramolecular complex of BDA with AG. MW irradiation of a water suspension of initial reagents dramatically reduced time for complex preparation in comparison with the conventional heating. The obtained BDA-AG supramolecular complex was used for preparation of water-soluble films. Acknowledgments: The work was partly supported by the RFBR (grant No. 16-33-50137) and by the Council for Grants of the President of RF for Support of Leading Scientific Schools (project No. NSh-8390.2016.3). References [1] S.A. Kuznetsova, B.N. Kuznetsov, E.S. Red’kina, V.A. Sokolenko, G.P. Skvortsova R.F. Patent

2324700, May 20, 2008. [2] S.A. Kuznetsova, G.P. Skvortsova, Yu.N. Malyar, N.Yu. Vasil’eva, B.N. Kuznetsov R.F. Patent

2469043, December 10, 2012. [3] S.A. Kuznetsova, B.N. Kuznetsov, A.G. Mikhailov, G.P. Skvortsova R.F. Patent 2273646, April 6,

2006. [4] S.A. Kuznetsova, T.P. Shakhtshneider, M.A. Mikhailenko, Yu.N. Malyar, V.V. Boldyrev R.F. Patent

2541153, December 04, 2013.

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Screening of Polysaccharides from Marine Microalgae and Study on its Antioxidant Activity

Zhan Jingting*,**, Huang Yongmei*,**, Yang Xiaohong*,**, Luo Hui*,**,#, Jiang Liming**,***,

*Chemistry Teaching and Research Section, Guangdong Medical University, Zhanjiang, 524023, China

**The Key Laboratory of Zhanjiang for R&D Marine Microorganism and Microalgae in the ***Institute of Biochemistry and Molecular Biology, Zhanjiang 524023, China

Beibu Gulf Rim, Zhanjiang 524023, China Corresponding author: Tel: 86-0759-2388523; [email protected]

#Tied for corresponding author: Tel: 86-0759-2388523; [email protected] Objective To screening for Polysaccharides marine microalga isolated from the intertidal zone of Naozhou Island (20°52′-20°56′N, 110°33′-110°38′E) and the Xuwen coral reef nature reserve (20°10′–20°27′N, 109°50′–109°24′E) in South China Sea and study on its antioxidant activity.

Methods

Polysaccharides-rich microalga were screening by a 96-well plate-based a modified

Anthrone-Sulfuric method. The antioxidant activity of polysaccharides was screened using

1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl-radical (OH) scavenging.

Results A total of 189 marine microalgal strains’ polysaccharides were determined by Anthrone-Sulfuric acid. Amongst, 21 strains of the isolated microalga were Polysaccharides-rich microalga, represented 22 species, distributed in Bacillariophyta,Rhodophyta,Haptophyta and Chlorophyta 4 phyla, 4 classes, 8 orders, 13 families, 11genera.The predominant genus of Polysaccharides-rich microalga was Amphora contained 6 strains, which takes a great proportion of 28.6 %. Conclusion The culturable Polysaccharides-rich microalga in the intertidal zone of Xuwen coral reef nature reserve was diversity, and would be the Ideal resources for research and development of Polysaccharides and biodiesel.

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The Extraction and Determination of Fatty Acids of Marine Microalgaes from Naozhou Island

Huang Yongmei*,**, Yang Xiaohong*,**, Zhan Jingting*,**, Xue Shanshan*,**, Luo Hui*,**,#, Jiang Liming**,***,

*Chemistry Teaching and Research Section, Guangdong Medical University, Zhanjiang, 524023, China

**The Key Laboratory of Zhanjiang for R&D Marine Microorganism and Microalgae in the ***Institute of Biochemistry and Molecular Biology, Zhanjiang 524023, China

Beibu Gulf Rim, Zhanjiang 524023, China Corresponding author: Tel: 86-0759-2388523; [email protected]

#Tied for corresponding author: Tel: 86-0759-2388523; [email protected]

Objective To extract fatty acids of Marine microalgaes and then determin their components. Methods The fatty acids were extracted with n-hexane, esterified with methyl esterification reagents, and then analyzed by GC-MS. Results The results of fatty acid analysis showed that the total lipids of 16 different kinds of microalgae be 0.6% to 13.38% of the microalgae dry weight, EPA be mainly existed in the Amphora and Nitzschia, and DHA be only detected in Isochrysis. Conclusion The culturable lipids-rich microalga from Naozhou Island was diversity, and would be the Ideal resources for research and development of lipids and biodiesel.

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A New Polymorph of Metacetamol – Promising Analogue of Paracetamol

Denis A. Rychkov1,2, Lindsay McGregor1,3, Paul L. Coster3, Sarah Day4, Valeri A. Drebushchak1,5, Andrei F. Achkasov1, Gary S. Nichol3,

Colin R. Pulham3, Elena V. Boldyreva2

1Novosibirsk State University, Pirogova, 2, Novosibirsk, Russia 2Institute of Solid State Chemistry and Mechanochemistry, Kutateladze, 18,

Novosibirsk, Russia 3School of Chemistry and Centre for Science at Extreme Conditions, The University of

Edinburgh, King's Buildings, David Brewster Road, Edinburgh, UK 4Diamond Light Source, Harwell Science & Innovation Campus, Didcot, UK

5V. S. Sobolev Institute of Geology and Mineralogy of the Russian Academy of Sciences, Pr. Ak. Koptyuga, 3, Novosibirsk, Russia

Polymorphism of molecular crystals is a well-known and very important area of research in materials science, and pharmaceutical research in particular. A change in crystalline structure can lead to different properties of polymorphs, wich can be beneficial for some properties of drug materials – solubility and dissolution dynamics, bioavailability and selectivity, etc. Paracetamol is well known example of diverse polymorphism of a widespread drug. Up to our best knowledge five polymorphs of paracetamol were discovered[1]. At the same time there is a discussion about high hepotoxicity of paracetamol in relation to its structural isomer metacetamol. It is surprising that prior to this work no polymorphs of metacetamol were found, resulting in only one crystal structure reported almost ten years ago.[2] In this study a new polymorph of metacetamol was discovered and investigated with variety of methods: IR, Raman and UV-Vis spectroscopy, single crystal and powder X-ray diffraction methods, thermal analysis including DSC and TG.[3] Structures of both polymorphs were compared and analyzed in relation to the paracetamol polymorphism phenomena described in the literature. Methods for producing single crystals and polycrystalline materials were developed, as well as usefull characteristics for new form were obtained – such as conditions for long shelf-life and for phase transitions between polymorphs. [3] This work was supported by the Year Abroad Programme of The University of Edinburgh, the Ministry of Education and Science of the Russian Federation project 1828. [1] S. J. Smith, M. M. Bishop, J. M. Montgomery, T. P. Hamilton, Y. K. Vohra, J. Phys. Chem. A. 118

(2014) 6068–77. [2] L. K. Hansen, G. L. Perlovich, A. Bauer-Brandl, Acta Crystallogr. Sect. E Struct. Reports Online 62

(2006) o3627–o3628. [3] L. McGregor, D. A. Rychkov, P. L. Coster, S. Day, V. A. Drebushchak, A. F. Achkasov, et al., Cryst.

Eng. Comm. 17 (2015) 6183–6192.

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Conformational Polymorphs of Furosemide Solvates

Alina A. Beloborodova*,**, Vasily S. Minkov**, Denis A. Rychkov*,**, Elena V. Boldyreva**

*Novosibirsk State University, Novosibirsk, Russia; **Institute of Solid State Chemistry and Mechanochemistry Siberian Branch

of the Russian Academy of Sciences

Polymorphism is very important for chemical sciences and technology. Usually, molecules with molecular fragments that are prone to rotation along a particular bond vector are found to crystallize in different conformational polymorphs, maintaining similar motifs in the crystal packing [1]. In contrast, polymorphs of small, rigid molecules differ in crystal packing only. Polymorphism of multicomponent crystals (MCC) is also neither new, nor rare. However, crystallization and the study of polymorphs of MCC is an emerging field. The existence of MCC that differ in the type or stoichiometry of the component molecules is well documented. At the same time, polymorphism of hydrate and solvate crystals, in which both the component species and the stoichiometries are identical but the crystal packings are different, are significantly less studied. Studying polymorphism of crystalline solvates is of great academic and industrial importance since different polymorphs have different properties and can yield different products on desolvation, including different polymorphs of single-component crystals. The present work describes and analyzes several polymorphs of solvates of furosemide with dimethylsulfoxide (DMSO) and dimethylformamide (DMF). Furosemide itself is a common loop diuretic drug for treating heart failure and edema. It has been studied extensively for a couple of decades. Having two molecular fragments exhibiting high rotational freedom along their respective covalent bonds, namely the furanylmethylamino and sulfamoyl fragments, furosemide can easily change its molecular conformation. In addition to our previous work [2], we report the crystal structures of solvates – furosemide-DMSO and furosemide-DMF, and analyze the relations between molecular conformations and molecular packing. Experimental work was complemented by DFT calculations. Number of theoretical and experimental techniques made it possible to rationalize the existence of conformational polymorphs of furosemide and relate molecular conformations, types of crystal structures formed and the conditions of their crystallization. The work was financially supported by the Ministry of Education and Science of the Russian Federation (Project 1828). [1] A. J. Cruz-Cabeza, J. Bernstein, Chem. Rev. 114 (2014) 2170–2191. doi:10.1021/cr400249d. [2] V. S. Minkov, A. A. Beloborodova, V. A. Drebushchak, E. V. Boldyreva, Cryst. Growth Des. 14

(2014) 513–522, doi:10.1021/cg401257w.

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Simple and Efficient Modifications of Well Known Techniques for Reliable Growth of High-Quality Crystals of Small Bioorganic Molecules

Sergey G. Arkhipov*,**, Denis A. Rychkov*,**, Elena V. Boldyreva**

*Novosibirsk State University, Pirogova, 2, Novosibirsk, Russia **Institute of Solid State Chemistry and Mechanochemistry, Kutateladze 18,

Novosibirsk, Russia

Polymorphism of molecular crystals is a very important issue for pharmaceutical science and industry, when such properties as solubility, shelf life, etc. should be double checked before release of the drug to the market. Thus, a complete description of all possible polymorphs is now recommended by Federal Food and Drug Administration (FDA) regulations [1]. To reveal, understand and control polymorphism of the system one is recommended to provide single crystal x-ray diffraction studies. For this reason, development of techniques for reliable growth of high-quality crystals of small molecules is almost mandatory nowadays. In this work we suggest a number of modifications to traditional techniques in order to overcome problems that frequently arise when growing crystals from solution. These improvements help to overcome following problems: poor nucleation, crystals adhering to the crystallization vessel or to each other, the spontaneous precipitation of many poor-quality small powder-like crystals, chemical degradation in the solution (e.g. Fig.1 A). Proposed techniques also can be used to crystallize desirable metastable polymorphs reliably, what is of extreme importance for pharmaceutical substances [2]. Examples are given, as well as precise description and photographs to learn these thechniques (e.g. Fig.1 B,C). Suggested improvements were used for reliable growth of small organic molecules such as amino acids, carboxylic acids, pharmaceuticals etc., but are definitely applicable to other classes of compound [2].

Fig. 1. The result of applying this modifications (A – before/B, C – after). Microphotographs B and C

demonstrating the removal of the crystallization solution [2]

This work was supported by the Ministry of Education and Science of the Russian Federation (project 1828)

[1] Federal Register, vol. 65, N. 251, 2000, pp. 83041–83063, [2] D.A. Rychkov, S.G. Arkhipov, E. V. Boldyreva, J. Appl. Crystallogr. 47 (2014) 1435–1442.

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The Glucuronidated and Sulfated Metabolites of Tilianin are Substrates of Breast Cancer Resistant Protein (ABCG2) in Mice

Qingwei Chen*, Liping Wang*,**, Xuejun Zeng*, Qiang Li**, Lijun Zhu**, Ming Hu**,***, Zhongqiu Liu*,**, Xinchun Wang*

*The First Affiliated Hospital of the Medical College, Shihezi University, Shihezi, Xinjiang, PR China

**International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, PR China

***College of Pharmacy, University of Houston, Houston, Texas, United States

The aim of this study was to investigate the role of breast cancer resistance protein (BCRP, ABCG2) in the disposition of tilianin (Til) and its metabolites in mice, Pharmacokinetic studies were carried out in Bcrp1(-/-) FVB mice compared with wild-type (WT) FVB mice. A sensitive UPLC-MS/MS method was developed to simultaneously quantify Til and its conjugate metabolites in mice plasma. Blood samples were collected at different time points within 24 h after intravenous administration (2 mg/kg) or oral administration (20 mg/kg, 10 mg/kg) of Til. The results showed that systemic exposure of Til in mice plasma were Til, acacetin (Aca), acacetin 5-glucuronide (Aca-5-G), acacetin 7-glucuronide (Aca-7-G) and acacetin 7-sulfate (Aca-7-S). The absolute bioavailabilities of Til were 2.15% and 2.57% after oral administration of Til at 20 mg/kg and 10 mg/kg in WT FVB mice, respectively. Compared with WT FVB mice, The Cmax and AUC values of Aca-7-G and Aca-7-S were significantly higher in Bcrp1(-/-) FVB mice (P<0.05). The oral bioavailabilities of Til were 0.73% and 0.08% after oral administration of Til at 20 mg/kg and 10 mg/kg, respectively. In conclusion, the applicability of UPLC-MS/MS assay was demonstrated and successfully applied for pharmacokinetics study of Til in WT FVB mice and Bcrp1 (-/-) FVB mice. Aca-7-G and Aca-7-S, rather than Til might be the substrates of BCRP. The interplay between phase II enzymes and BCRP leads to extensive metabolism and low bioavailability for Til in mice.

Acknowledgements：We thank the support from the joint fund set up by the National Natural Science foundation of China and the Government of the Xinjiang Uygur Autonomous Region (Grant U1203204), the Key International Joint Research Project of National Natural Science Foundation of China (Grant 81120108025).

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Synthesis and Validation of (R)-3,3,3-trifluoro-2-hydroxy-2-Methyl-propionamides as Pyruvate Dehydrogenase Kinase Inhibitors

To Reduce the Growth of Cancer Cells

Shao-Lin Zhang, Wen Zhang, Zheng Yang, Xiaohui Hu, Kin Yip Tam

Drug Development Core, Faculty of Health Sciences, University of Macau, Macau, China [email protected]

Most cancer cells exhibit a high rate of glycolysis and reduced capacity in mitochondrial oxidative phosphorylation. The expression of pyruvate dehydrogenase kinases (PDKs) was found to be increased in many cancer cells. Inhibition of PDKs could disrupt the balance between the demand and supply of oxygen, resulting in cancer cell death. Recently, PDKs have received considerable attention as a promising anticancer target [1, 2]. In 1999, Aicher from Novartis reported that (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamides could inhibit PDKs in primary enzymatic assay, and decrease the lactate formation in vivo [3]. However, the use of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamides as PDK inhibitor to reduce the growth of cancer cells has not been reported in open literature. Herein, we report the synthesis and validation of novel PDK inhibitors 1 and 2 (Fig. 1) containing (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide moiety, which reduce the growth of cancer cell lines in vitro.

Inhibition the growth of cancer cell, % a

Comp. ID RKO U251 SF188 T98G RES259 T47D

1 88.6 82.0 82.0 67.8 77.6 71.0 2 90.1 59.5 77.3 34.7 46.8 68.7

AZD7545 16.3 1.9 12.3 -1.2 0.08 29.2 a compounds at 40 µM against the growth of different cancer cell lines.

Fig. 1. Chemical structures of the synthesized compounds and their inhibition on different types of cancer cell lines

Reference [1] S. L. Zhang, X. H. Hu, W. Zhang, K. Y. Tam, J. Med. Chem. 59 (2016) 3562-3568. [2] S. L. Zhang, X. H. Hu, W. Zhang, H. K. Yao, K.Y. Tam, Drug Discov. Today 20 (2015) 1112-1119. [3] T. D. Aicher, R. C. Anderson, G. R. Bebernitz, G. M. Coppola, C. F. Jewell, et al, J. Med. Chem. 42

(1999) 2741-2746.

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Screening for Culturable Marine Bacteria Producing Exopolysaccharide from the Intertidal Zone of Zhanjiang Coast

Shan-Shan Xue*,**, Ying-Wen Liu*,**, Li-Jiao Cui*,**, Yong-Mei Huang**,***, Li-Ming Jiang*,**,

*Institute of Biochemistry and Molecular Biology, Guangdong Medical University,Guangdong 524023,China

**Micro-Algae and Micro-Organisms Resources in Beibu Gulf Coast Research Laboratory, Guangdong Medical University, Guangdong 524023,China

***Department of Medical Laboratory, Guangdong Medical University, Guangdong 524023,China

Corresponding author. Tel: 86-0759-2388523; [email protected] Objective To Screening for culturable marine bacteria producing exopolysaccharide(EPS) from the intertidal zone of Naozhou Island (20°52′–20°56′N, 110°33′–110°38′E) and Xuwen coral reef nature reserve (20°10′–20°27′N, 109°50′–109°24′E) in the South China Sea. Methods The samples of both seawater and sediment were collected from the intertidal zone of Naozhou Island and Xuwen coral reef nature reserve in South China Sea, and bacteria strains were isolated from the samples by conventional culture-dependent method, and then the isolated strains were investigated by a phylogenetic analysis based on the 16S rRNA gene sequences, screening for the bacterial strains producing exopolysaccharide(EPS) by Sulfuric-anthrone methods. Results A total of 94 bacterial strains were isolated, purified and collected from the samples on the plates of LB dextrose agar medium prepared with 50% seawater, identified successfully by PCR amplification, sequencing, BLAST and phylogenetic analysis of the 16S rRNA gene sequences. These 94 strains represented 51 species, belonging to 20 genera of 14 families in 10 orders, 3 classes, spreading in two phyla, Firmicute and Proteobacteria. The predominant genus was Bacillus, which takes a great proportion of 37.50 %, followed by Vibrio, which takes a proportion of 18.75 %. Conclusion A total of 94 marine bacterial strains were isolated, purified, collected and identified, in which, 53 strains belonged to 22 species distributed in 7 genera could produce extracellular proteases, and the predominant genus is Bacillus, followed by Exiguobacterium. 27 strains belonged to 17 species distributed in 3 genera could produce exopolysaccharide (EPS) highly and the predominant genus was Bacillus, followed by Exiguobacterium. This work was supported by the grants from Zhanjiang Municipal Governmental Specific Financial Fund Allocated for Competitive Scientific and Technological Projects, the municipal key laboratory construction project(2012E02).

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Study on Screening of Oil-Producing Bacteria in Five Methods

Ying-Wen Liu*,**, Shan-Shan Xue*,**, Li-Jiao Cui*,**, Yong-Mei Huang**,***, Li-Ming Jiang*,**,

*Institute of Biochemistry and Molecular Biology, Guangdong Medical University,Guangdong 524023,China

**Micro-Algae and Micro-Organisms Resources in Beibu Gulf Coast Research Laboratory, Guangdong Medical University, Guangdong 524023,China

***Department of Medical Laboratory, Guangdong Medical University, Guangdong 524023,China

Corresponding author. Tel: 86-0759-2388523; [email protected] Objective To find a simple and rapid method to screen the marine bacteria and determine the intracellular lipid content. Methods This paper mainly discusses five method of screening oil-producing bacteria that were H₂O₂-plate assay, TTC (triphenyltetrazolium chlorid) reduction assay and three lipid dyes assay (Sudan black B, Nile red, BODIPY). With the 94 Bacteria strain that have been sequenced strains for experiments, using intuitive quick-Plate Method to screen the oil-producing bacteria. Results Based on the Nile red -plate, it is showed that 27 of 94 strains are oil-producing. The confirmation of assay result was performed by injecting fatty acid methyl esters produced by the selected strains to Gas Chromatography - Mass Spectrometer (GCMS). To date, this assay is the most effective, inexpensive and specific method for bacteria producing fatty acid. Conclusion Nile red is fluorescent dye that lipid combined with it would deliver orange red fluorescent under UV light. The Sudan B is not a fluorescent dye, and we can be seen no special change in white light. Bacteria that producing EPA reduce TTC to red TF. BODIPY is a new fluorescent dye, but it fades easily and it is expensive. The oxidative stability of fatty acid in growing bacteria towards added H2O2 is a characteristic between the fatty acid producers (no zone of inhibition) and no-fatty acid producers (zone of inhibition) by direct visualization with a high error rate. To sum up, the method with Nile red offer a rapid, inexpensive and intuitive tool to screen oil-producing bacteria. This work was supported by the grants from Zhanjiang Municipal Governmental Specific Financial Fund Allocated for Competitive Scientific and Technological Projects, the municipal key laboratory construction project(2012E02).

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SLM Attenuates Apoptosis Induced by Amyloid Beta 1-42 in SH-SY5Y Human Neuroblastoma Cells

Xiaoli Wu, Jayasankar Kosaraju, Xiaohui Hu, Kin Yip Tam

Drug Development Core, Faculty of Health Sciences, University of Macau, Macau, China E-mail: [email protected]

Alzheimer's disease (AD) is a neurodegenerative disorder accumulated with numerous deposits of amyloid beta (Aβ) aggregates in the patient brain. Clearance or prevention of forming Aβ aggregates is a promising therapeutic approach for the treatment of AD. SLM is a carbozole-based cyanine fluorophore compound that binds to Aβ and inhibits its aggregation leading to neuronal toxicity in AD [1]. In the present study we investigate the effect of SLM on toxicity induced by Aβ42 in cultured SH-SY5Y neuroblastoma cells. Treatment of SH-SY5Y cells with Aβ42 induced toxicity was confirmed by reduced cell viability and increased apoptosis. Pretreatment of the cells with SLM enhanced cell viability and attenuated the apoptosis induced by Aβ42. From our data we hypothesize that the anti-apoptosis activity is dependent on the regulation of the AKT/GSK3β signaling pathway. Altogether, the present study suggests that SLM suppresses the stimulation of caspase cascades and may have potential in the prevention of Aβ-mediated diseases, such as AD. Reference [1] W. Yang, Y. Wong, O.T. Ng, L.P. Bai, D.W. Kwong, Y. Ke, Z.H. Jiang, H.W. Li, K.K. Yung, M.S. Wong,

Angew Chem. Int. Ed. Engl. 51(8) (2012) 1804-10.

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The Effects of Icariin on the Macrophage Apoptosis Induced by Ox-LDL and the Underlying Mechanisms

Luo Juan*,**, Ji Yanqiong***, Tan Yan*, Pan Longrui*, Wang Xuanbin**, Li Hongliang**, Zhang Qiufang*,**

*Hubei Key Laboratory of Wudang Local Chinese Medicine Research **Department of Pharmacology, Hubei University of Medicine

***Department of Cardiology, Taihe Hospital, Shiyan 442000, China

AIM To investigate the inbitory effects of icariin(ICA) on oxidized low density lipoprotein(oxLDL)-induced macrophage apoptosis and the underlying mechanisms. METHODS RAW264.7 macrophages were pretreated with ICA (10, 20, 40μmol/L) or 5 mmol/L 4-phenylbutyric acid (4-PBA) for 1 h. Then treated with oxLDL (100 mg/L) for 24h, the cell viability and apoptosis were detected by MTT assay and annexin-FITC/PI double staining method, respectively. Intracellular lipid droplets were assayed by oil red O staining, caspase-3 activity was measured by chromatometry, endoplasmic reticulum stress markers (GRP78) was detected by Western blot. RESULTS Pretreatment with ICA (20, 40 μmol/L) could protect macrophages from injury induced by oxLDL, as assessed by the increased cell viability and the decreased apoptotic rate and significantly attenuated lipid accumulation in RAW264.7 cells. Compared with oxLDL group, the activity of capase-3 and the protein level of GRP78 in the macrophages were decreased in ICA preteated group. CONCULSIONS: ICA can prevent macrophages damage induced by oxLDL, the mechanisms may partially involve its ability to decrease the activity of caspase-3 and down-regulated ERS.

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Tanezumab for Patients with Osteoarthritis of the Knee: A Meta-Analysis

Shun-Li Kan, Yan Li, Guang-Zhi Ning, Ling-Xiao Chen, Shi-Qing Feng

*Department of Orthopaedics, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin, China

Objective: Tanezumab is a new therapeutic intervention for patients with osteoarthritis (OA) of the knee. We performed the present meta-analysis to appraise the efficacy and safety of tanezumab for patients with knee OA. Methods: We systematically searched randomized controlled trials from PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL). The primary outcomes were mean change in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) Pain, WOMAC Physical Function and patient's global assessment (PGA). Outcomes were reported as standard mean difference (SMD) or relative risk (RR) with 95 % confidence interval (CI). We assessed the pooled data using a random-effects model. Results: Of the identified studies, four were eligible and were included in this meta-analysis (N=1839 participants). Compared with the placebo groups, tanezumab yielded a significant reduction in mean change in WOMAC Pain (SMD=0.51, 95 % CI 0.34 to 0.69, P<0.00001), WOMAC Physical Function (SMD=0.56, 95 % CI 0.38 to 0.74, P<0.00001) and PGA (SMD=0.34, 95 % CI 0.22 to 0.47, P<0.00001). There was no significant difference in serious adverse events (RR=1.06, 95 % CI 0.59 to 1.92, P=0.84) between the tanezumab and placebo groups. Tanezumab significantly increased discontinuations due to adverse events (RR=2.89, 95% CI 1.59 to 5.26, P=0.0005), abnormal peripheral sensations (RR=3.14, 95 % CI 2.12 to 4.66, P<0.00001), and peripheral neuropathy (RR = 6.05, 95 % CI 2.32 to 15.81, P=0.0002). Conclusion: Tanezumab can alleviate pain and improve function for patients with OA of the knee. However, considering the limited number of studies, the conclusion should be interpreted cautiously and more clinical randomized controlled trials are needed to verify the efficacy and safety of tanezumab for OA of the knee.

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Author Index

A Achkasov, AF ............................................... 40 Allah Mohd Ali, GK ...................................... 35 Alvebratt, C ................................................... 7 Arba, M ................................................. 23, 24 Arkhipov, SG ............................................... 42 Avdeef, A ................................................ 9, 20

B Beloborodova, AA ....................................... 41 Bergström, CAS ....................................... 7, 13 Boldyreva, EV ............................ 11, 40, 41, 42

C Chen, C-S ..................................................... 18 Chen, H ....................................................... 15 Chen, L-X ..................................................... 49 Chen, Q ....................................................... 43 Chen, T ........................................................ 17 Chen, X ........................................................ 14 Coster, PL .................................................... 40 Cui, L-J ................................................... 45, 46

D Day, S .......................................................... 40 Drebushchak, VA ........................................ 40 Du, S ............................................................ 10 Dulski, M ..................................................... 12

E Edueng, K .................................................... 13 Elhadi, N ...................................................... 35 Elnabi, NJ .................................................... 35

F Feng, J ......................................................... 14 Feng, S-Q ..................................................... 49

G Gadabadze, M ............................................. 34 Gao, W .......................................................... 6 Gong, J ........................................................ 10 Gorgoshidze, M ........................................... 34 Guo, L .......................................................... 33

H Hilfiker, R ....................................................... 8 Hongliang, L ................................................ 48 Hu, M .......................................................... 43 Hu, X ................................................ 16, 44, 47 Huang, Y-C ................................................... 18 Huang, Y-M ........................................... 45, 46 Hui, L ..................................................... 38, 39

J Jiang, B ........................................................ 17 Jiang, L-M .............................................. 45, 46 Jingting, Z .............................................. 38, 39 Juan, L ......................................................... 48

K Kan, S-L ....................................................... 49 Kiladze, M ................................................... 34 Knapik, J ...................................................... 26 Kosaraju, J ................................................... 47 Kuo, S-C ....................................................... 18 Kuznetsova, SA ...................................... 36, 37 Kwok, HF ..................................................... 28


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L Li, C ............................................................. 15 Li, J ................................................................ 3 Li, Q ............................................................. 43 Li, T ............................................................... 6 Li, X ................................................. 14, 31, 32 Li, Y ............................................................. 49 Liao, W-Y ..................................................... 18 Liaw, C-C ..................................................... 18 Liming, J ................................................ 38, 39 Liu, J ............................................................ 33 Liu, Y ........................................................... 15 Liu, Y-W ................................................. 45, 46 Liu, Z ........................................................... 43 Longrui, P .................................................... 48 Lou, L .......................................................... 14

M Ma, Z ........................................................... 15 Mahdi, ES .................................................... 35 Mahlin, D .................................................... 13 Malyar, YN .................................................. 37 Maran, U ..................................................... 22 McGregor, L ................................................ 40 Mikhailenko, MA ........................................ 37 Minkov, VS .................................................. 41 Mo, Z ........................................................... 15 Monaselidze, J ............................................ 34 Murphy, G ................................................... 28 Myz, SA ....................................................... 36

N Ning, G-Z ..................................................... 49

O Ogienko, AA ................................................ 36 Ogienko, AG ................................................ 36 Oja, M ......................................................... 22

P Paluch, M .............................................. 12, 26 Pankrushina, NA ......................................... 37 Peters, GJ ...................................................... 4 Petersson, E .................................................. 7 Pulham, CR.................................................. 40

Q Qiufang, Z .................................................... 48

R Rams-Baron, M ........................................... 12 Ruslin .................................................... 23, 24 Rychkov, DA .......................................... 40, 41

S Serajuddin, ATM ......................................... 25 Shakhtshneider, TP ............................... 36, 37 Shanshan, X ................................................. 39 Shen, C-N .................................................... 18 Strömme, M .................................................. 7 Su, Z ............................................................ 15 Sugano, K(K) ................................................ 27 Sutrisna, R ............................................. 23, 24

T Tam, KY ........................................... 16, 44, 47 Tan, L ........................................................... 15 Tao, W ......................................................... 14 Tian, Y ......................................................... 19

V Valko, K ....................................................... 21

W Wan, H ........................................................ 14 Wang, J ........................................................ 10 Wang, L ....................................................... 43 Wang, X ....................................................... 43 Wang, Y ....................................................... 10 Wang, Z ....................................................... 19 Włodarczyk, P ............................................. 12 Wojnarowska, Ż .......................................... 12 Wong, K-Y ..................................................... 5 Wu, X ........................................................... 47

X Xia, Y ........................................................... 17 Xiaohong, Y ........................................... 38, 39 Xie, J ............................................................ 15 Xie, R ........................................................... 28 Xu, W ........................................................... 19


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Xu, Y ...................................................... 15, 17 Xuanbin, W ................................................. 48 Xue, S-S ................................................. 45, 46

Y Yan, T .......................................................... 48 Yang, C ........................................................ 14 Yang, L ........................................................ 19 Yang, X ........................................................ 15 Yang, Y ........................................................ 17 Yang, Z ........................................................ 44 Yanqiong, J .................................................. 48 Ye, J ............................................................. 28 Yin, Z ........................................................... 33 Yongmei, H ........................................... 38, 39 Yuen, SM ..................................................... 28

Z Zamay, AS.................................................... 37 Zeng, X ........................................................ 43 Zhang, G ...................................................... 14 Zhang, Ha .................................................... 19 Zhang, Hu .................................................... 19 Zhang, J ................................................. 31, 32 Zhang, L ....................................................... 14 Zhang, S-L .............................................. 16, 44 Zhang, W ............................................... 16, 44 Zheng, S ....................................................... 19 Zheng, X ...................................................... 33 Zhi, F ............................................................ 17 Zhong, D ...................................................... 14 Zhou, J ......................................................... 15 Zhu, L ........................................................... 43

3rd World Conference on Physico-Chemical Methods in Drug Discovery and Development

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