MEAT AND MILK PRODUCTS OF DAIRY COWS AS RISK FACTORS FOR CANCERS AND NEUROLOGICAL DISEASES

Harald zur Hausen
Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Abstract

Slightly more than 20% of the global cancer incidence is linked to viral, bacterial, or parasitic infections. Their discovery permitted novel approaches to cancer prevention either by vaccination (Hepatitis B and human papillomaviruses) or by therapeutic interference with persistent infections (HIV, Hepatitis C, Helicobacter pylori, Schistosoma and liver flukes). These developments encourage further research on a potential involvement of other infections in cancers, not yet linked to exogenous agents. We presently study the putative role of novel viruses in cancers of the colon and the breast, but also in multiple sclerosis (MS). Epidemiological studies suggested a possible role of infectious events in these malignancies and MS, probably due to consumption of bovine infected meat and dairy products. A number of novel single-stranded DNAs have been isolated from bovine serum and milk, but also from individual multiple sclerosis lesions. They are presently being analysed for a possible role in these cancers and neurological diseases.

ANTI-CANCER DRUG DISCOVERY AND THERAPY

Attilio Di Pietro
BMSSI UMR5086 CNRS-University of Lyon,
Institute of Protein Biology and Chemistry,
Passage du Vercors 7, 69367 Lyon, France.
Email: a.dipietro@ibcp.fr

ANTI-CANCER DRUG DISCOVERY AND THERAPY

Attilio Di Pietro
BMSSI UMR5086 CNRS-University of Lyon,
Institute of Protein Biology and Chemistry,
Passage du Vercors 7, 69367 Lyon, France.
Email: a.dipietro@ibcp.fr

Academic CRO/Industrial Collaborations in Drug Discovery  (Track)

NUCLEIC ACID-BASED DRUGS AGAINST EMERGING ZOONOTIC VIRUSES

Jonathan P. Wong
Defence R&D Canada, Suffield Research Centre, Ralston, Alberta, Canada.

Abstract

Global outbreaks of diseases caused by zoonotic viruses, including Ebola, MERS, SARS, avian influenza and others, have steadily increased in recent years. Emerging zoonotic viruses are generally phylogenetically diverse, are unpredictable and are known to cause diseases with high case fatality rates in humans and are hard to protect against due to lack of approved antiviral drugs and vaccines. The aim of this review is to discuss how advances in genomics, rational drug design and innate immune signaling can contribute to the design of nucleic acid-based drugs which can play a significant role in combating these emerging threats. Nucleic acid-based drugs (NABDs) are increasingly being recognized as promising antiviral therapeutic agents due to their versatile mechanisms of action, safety and efficacy profiles in preclinical studies, and amenable to address drug resistance arising from viral mutations. Specifically, the antiviral activity of siRNAs, antisense oligonucleotides and microRNAs is mediated by sequence-specific gene-silencing, and broad-spectrum innate and antiviral immune responses can be elicited by toll-like receptor agonists as such double-stranded RNA and CpG oligonucleotides. The purpose of this lecture is to highlight selective classes of NABDs and to discuss their mechanisms of action, preclinical safety and efficacy studies, current state of development, and provide future perspectives on the overall development of NABDs against emerging zoonotic viral diseases. In addition, challenges and barriers to the clinical development of NABDs and drug development against rare and neglected zoonotic viral diseases will also be highlighted in this review. In summary, NABDs are well suited to address emerging zoonotic viral diseases because they can be designed with great flexibility to match prevalent strains of zoonotic viruses, are robust and stable in many environmental conditions, and are versatile in mechanism of action to address viral mutations and drug resistance.

Anti-Cancer Drub Discovery & Therapy (Track)

SYNERGISM FROM COMBINATION OF TARGETED THERAPY WITH TUMOUR ACTIVE PHYTOCHEMICALS IN OVARIAN TUMOUR MODELS AND CHANGES IN PROTEIN EXPRESSION

Fazlul Huq

Discipline of Biomedical Science Sydney Medical School, University of Sydney, Australia

Ovarian cancer is most lethal cause of gynecological types of cancer mainly because of acquired drug resistance and poor diagnosisis so that in majority of cases it is detected at an advanced stage when it is likely to have spread beyond ovaries. Combination of platinum drugs with paclitaxel is the current mode of treatment. When the cancer cells respond well to the drug combination, relapse often occurs for which drugs fail to function. The present study aimed to investigate synergism from combination of platinum drugs and designed platinums with tumour active phytochemicals including curcumin, EGCG, thymoquinone, resveratrol, ursolic acid and genistein in human ovarian tumour models. Activity of the compounds alone and in sequenced combination in ovarian cancer cell lines including A2780, A2780cisR and A2780ZD0473R were determined using MTT reduction assay. Drug uptake and drug-DNA binding were determined using developed protocols. Proteomic studies involving 2D gel electrophoresis and mass spectrometry were carried out to characterize key proteins associated with platinum resistance.

Generally but not always sequenced combinations with 2 to 4 h time gap were found to be synergistic. The variation in combined drug action with the change in sequence of administration clearly indicates that the action of one drug has modulated that of the other. Proteomic studies have identified over thirty proteins believed to be associated with platinum resistance in ovarian cancer. They belong to six major groups including cytoskeletal proteins, molecular chaperone and stress related proteins, proteins involved in detoxification and drug resistance, proteins involved in metabolic processes and mRNA processing proteins. The proteins are restored to normalcy due to treatment with synergistic combinations. If confirmed in vivo, the results of the study suggest that appropriate combinations of targeted therapy and tumour active phytochemicals may providing an effective and affordable means of overcoming drug resistance in ovarian cancer.

Anti-Infectives (Track)

SELECTIVE ER TRANSLOCATION INHIBITORS AS NOVEL ANTIVIRAL DRUGS

Vermeire Kurt

Rega Institute for Medical Research, KU Leuven, Leuven, Belgium;

Successful virus entry in target cells begins with attachment of the virus to the cell surface. Each virus has its set of viral glycoproteins that bind to specific cellular surface membrane proteins. A wellknown example is the CD4 molecule on T-helper cells that serves as the main receptor for the Human Immunodeficiency Virus (HIV). In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP) and targets the ribosome-nascent chain complex (RNC) to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, signal peptides have diverse but unique primary sequences. Thus, drugs that recognize signal peptides could be exploited to inhibit translocation of specific proteins into the ER. The small-molecule drug cyclotriazadisulfonamide (CADA) was previously selected for its anti-HIV activity and identified as a highly selective human CD4 (hCD4) down-modulator. Here, we show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal signal peptide of hCD4. Moreover, through surface plasmon resonance (SPR) analysis we could show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. These findings demonstrate that a synthetic, cell-permeable smallmolecule can be developed as a signal peptide-binding drug to selectively and reversibly inhibit protein translocation. This drug-inducible regulation of the expression of specific cellular surface proteins involved in virus entry could open new roads to the successful development of novel antiviral therapeutics.

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Cardiovascular Drug Discovery and Therapy  (Track)

CYTOKINES AND ATHEROSCLEROSIS: MOLECULAR MECHANISMS AND THERAPEUTIC TARGETS

Thomas S. Davies, Hayley Gallagher, Joe W. E. Moss, Wijdan Al-Ahmadi and Dipak P. Ramji

Cardiff School of Biosciences, Cardiff University, Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK;

Atherosclerosis, an inflammatory disorder of the vasculature and the underlying cause of myocardial infarction and cerebrovascular accident, is responsible for most global morbidity and mortality. The inflammatory response in atherosclerosis is orchestrated by cytokines, which regulate all stages of the disease and therefore represent promising therapeutic targets. Approaches to attenuate proinflammatory cytokine actions include neutralization via blocking antibodies or soluble decoy receptors, receptor antagonists and small molecule inhibitors of intracellular signaling pathways. Other avenues include use of antiinflammatory cytokines or agents that augment their actions. An in-depth understanding of cytokine actions in atherosclerosis is necessary in order to validate their therapeutic potential and to identify potentially new therapeutic targets/approaches.

We have been investigating cytokine actions and signaling in atherosclerosis for several years with focus on macrophages, which play pivotal roles in all stages of the disease. Our research has provided novel insights into the effects, intracellular signaling pathways and underlying molecular mechanisms by which the cytokines interferongamma, transforming growth factor-beta, tumour necrosis factor-like protein 1A and interleukin-33 modulate atherosclerosis and key macrophage processes such as foam cell formation, gene expression and control of the inflammatory response. These will be presented in the context of current therapies and future drug discovery and development.

Ramji, D. P. and Davies, T. S. (2015) Cytokines in atherosclerosis: Key players in all stages of disease and promising therapeutic targets. Cytokine Growth Factor Reviews. DOI: 10.1016/j.cytogfr.2015.04.003.

Buckley, M. L. and Ramji, D. P. (2015) The influence of dysfunctional signaling and lipid homeostasis in mediating the inflammatory responses during atherosclerosis. Biochimica et Biophysica Acta-Molecular Basis of Disease 1852:1498-1510.

McLaren, J. E., Michael, D. R., Ashlin, T. G. and Ramji, D. P. (2011) Cytokines, macrophage lipid metabolism and foam cells: Implications for cardiovascular disease therapy. Progress in Lipid Research. 50:331-337.

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Cardiovascular Drug Discovery and Therapy  (Track)

MEDICAL MANAGEMENT OF THE PATIENT WITH DIABETES AND PAD

Fadi Shamoun

Medical Director, NP Cardiology Fellowship, Medical Director, Anticoagulation Clinic, Assistant Professor of Medicine, Vascular Medicine, Division of Cardiovascular Diseases;

Peripheral arterial disease (PAD) caused mostly be atherosclerosis of the lower extremity arteries affects 8-9 million Americans and more than 200 million worldwide. The prevalence of PAD increased 23% over the past 10 years due to longer life expectancy and the epidemic of obesity and diabetes.

PAD is manifested by a variety of lower extremity symptoms ranging from atypical leg pain to classic walking limiting claudication. Diabetes could lead to peripheral neuropathy and lower extremity symptoms as well.

Diabetic patients, especially with poorly controlled diabetes, could progress rapidly from mild PAD to acute limb ischemia and subsequently amputation. Diabetic patients are at an elevated risk for disease recurrence and complication post angioplasty. Medical therapy for PAD includes adequate foot care, walking program, adjunctive therapy and optimal blood sugar. The effect of newer diabetic medication on lower extremity arterial disease is still unclear.

Chemistry  (Track)

DISCOVERING NEW APPLICATIONS OF CHIRAL N-SULFINYL IMINES IN ASYMMETRIC SYNTHESIS

Miguel Yus

Departamento de Química Orgánica and Centro de Innovación en Química Avanzada (ORFEOCINQA), Universidad de Alicante, Apdo. 99, 03080 Alicante, Spain;

Chiral N-sulfinyl imines 1, especially the corresponding N-tert-butyl substituted derivatives are interesting starting materials in asymmetric synthesis because (a) they are easily accessible in both enantiomerically pure form, (b) the sulfinyl group activates the imine moiety towards nucleophilic substitution so, in the reaction with different nucleophiles an asymmetric induction takes place giving an diastereoenriched product, which can be easily separated into the corresponding pure diastereomers, and (c) the deprotection of the amino group, after the addition of the nucleophile can be easily achieved by simple treatment with hydrochloric acid. In this presentation, the reactivity and synthetic applications of these materials in the rutheniumcatalyzed hydrogen transfer, addition of alkyl zincates and indium-promoted allylation (to give chiral amines 2) will be considered. Especial attention is paid to the synthetic applications of the mentioned processes, mainly for the preparation of natural or unnatural alkaloids and amino acids [1, 2].

[1]. For reviews, see: (a) Foubelo, F.; Yus, M. Eur. J. Org. Chem. 2014, 485-491. (b) Yus, M.; González-Gómez, J. C.; Yus, M. Chem. Rev. 2013, 113, 5595-5698. (c) Yus, M.; González-Gómez, J. C.; Yus, M. Chem. Rev. 2011, 111, 7774-7854. For last papers on this topic from our group, see: (d) García-Muñoz, M. J.; Foubelo, F.; Yus, M. Heterocycles 2015, 90, 1419-1432. (e) Barros, O. S. R.; Sirvent, J. A.; Foubelo, F.; Yus, M Chem. Commun. 2014, 50, 6898-6901. (f) García-Muñoz, M. J.; Dema, H. K.; Foubelo, F.; Yus, M. Tetrahedron: Asymmetry 2014, 25, 362-372. (g) Sirvent, J. A.; Foubelo, F.; Yus, M. J. Org. Chem. 2014, 79, 1356- 1367. (h) Sirvent, J. A.; Foubelo, F.; Yus, M. Heterocycles 2014, 88, 1163-1174. (i) Sirvent, J. A.; Foubelo, F.; Yus, M. Eur. J. Org. Chem . 2013, 2461-2471. (j) García-Muñoz, M. J.; Zacconi, F.; Foubelo, F.; Yus, M. Eur. J. Org. Chem. 2013, 1287-1295.

[2]. I thank the Spanish Ministerio de Economía y Competitividad (CTQ2011-241665, CTQ2014-53695-P, and CTQ2014-51912- RED) and the Generalitat Valenciana (PROMETEO 2014/017) for their generous financial support.

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Chemistry  (Track)

DUAL ENANTIOSELECTIVE CONTROL IN ASYMMETRIC SYNTHESIS

So Won Youn, Sam Min Kim, Doo Han Park and Yong Hae Kim

Center for Molecular Design and Synthesis, Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Taejon 305-701, S. Korea

Stereo-differentiated asymmetric syntheses have been achieved by multifunctionalizations of S-β- amino alcohol ligands. Diels-Alder cycloadditions of S-indoline chiral acrylamides with cyclopentadiene or butadiene proceed with high diastereofacial selectivity and enantiomeric excess, giving either endo-R to endo-S products depending of Lewis acid and the structures of chiral dienophiles. Diastereo-and enantio-selective pinacol coupling reactions of α-ketoamides mediated by samarium diiodide afforded extremely high diastereoselectivities. Enantiopure (S,S)- (up to > 99.5%ee) or (R,R)- (up to > 99%ee) 2,3- dialkyltartaric acid and derivatives can be synthesized for the first time depending on the structures of α-ketoamides [1].

[1] S. M. Kim, I. S. Byun, and Y. H. Kim, Angew. Chem. Int. Ed. 2000, 39, 728.; Y. H. Kim, Acc. Chem. Res. 2001, 34, 955.

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Chemistry  (Track)

FREE RADICAL THERAPEUTICS

Carl H. Schiesser

School of Chemistry, The University of Melbourne, Victoria, 3010, Australia

Free radicals are important entities that are associated with a range of biological signalling events. Utilising free radicals, or molecules that react with free radicals, to interfere with these signalling processes can have important, therapeutically-useful outcomes in areas ranging from skin-tissue repair to the control of bacterial biofilms. This presentation will provide examples of our recent work in these areas.

Diabetes is Australia’s fastest growing epidemic with one person diagnosed every 5 minutes. Research has shown that diabetic patients in hospital have increased risk of mortality and further complications compared to non-diabetic patients. Furthermore, individuals who develop a diabetic foot ulcer (DFU) as a result of poor glycaemic management and inadequate foot care are at greater risk of premature death, myocardial infarction and fatal stroke than those without a history of DFU. We have shown that 1,4-anhydro-4-seleno-D-talitol (1), is a remarkable compound that accelerates tissue repair and wound healing and is particularly effective in repairing diabetic wounds [1].

Bacterial biofilms are notoriously problematic in areas such as medicine, food, water treatment and cultural heritage conservation. These surface-associated assemblages of microorganisms are refractory to many traditional treatments, such as antibiotics and biocides.

We have shown that 3-(dodecane-1-thiyl)-4-(hydroxymethyl)-2,2,5,5-tetramethyl -1-pyrrolinoxyl (2) significantly suppresses biofilm formation and elicits dispersal events in both P. aeruginosa and in mixed-culture biofilms [2].

[1] Storkey, C.; Davies, M. J.; Schiesser, C. H. Int. Patent Appl. PCT/AU2014/000960.

[2] Alexander, S.-A.; Rouse, E. M.; White, J. M.; Tse, N.; Kyi, C.; Schiesser, C. H. Chem. Commun., 2015, 51, 3355.

Chemistry  (Track)

NEW PYRANO [2, 3-D: 6, 5-D''] DIPYRIMIDINE DERIVATIVES - SYNTHESIS, IN VITRO CYTOTOXICITY ACTIVITY AND COMPUTATIONAL STUDIES

Suresh Maddila, Sridevi Gorle, Moganavelli Singh and Sreekantha B. Jonnalagadda

Department of Chemistry, School of Chemistry & Physics, University of KwaZulu-Natal, Durban, South Africa;

A series of new pyrano [2, 3-d: 6, 5-d''] dipyrimidine derivatives have been synthesized and evaluated for their in vitro anticancer activity. The structure of all the synthesized compounds was confirmed by 1H NMR, 13C NMR, 15N NMR, HR-MS and FT-IR spectral analysis. These compounds were studied for cytotoxic activity against four human cancer (HeLa, SkBr3, HepG2, and Caco2) cell lines. The synthesized compounds showed high selectivity and four compounds (5e, 5f, 5g and 5i) showed excellent potent cytotoxicity against HeLa, SkBr3, and Hep-G2 cancer cell lines. Furthermore, four compounds (5a, 5c, 5b and 5d) have exhibited significant cytotoxicity activity against for SkBr3 and HepG2 cell lines and moderate cytotoxicity against HeLa cell line. Virtual screening was performed via docking to determine the binding modes of synthesized compounds into the binding site of Human Kinesin Eg5.

Synthesis, pyrimidines, pyranes, cytotoxicity activity, computational studies.

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Email: Email

Academic CRO/Industrial Collaborations in Drug Discovery  (Track)

NUCLEIC ACID-BASED DRUGS AGAINST EMERGING ZOONOTIC VIRUSES

Jonathan P. Wong
Defence R&D Canada, Suffield Research Centre, Ralston, Alberta, Canada.

Abstract

Global outbreaks of diseases caused by zoonotic viruses, including Ebola, MERS, SARS, avian influenza and others, have steadily increased in recent years. Emerging zoonotic viruses are generally phylogenetically diverse, are unpredictable and are known to cause diseases with high case fatality rates in humans and are hard to protect against due to lack of approved antiviral drugs and vaccines. The aim of this review is to discuss how advances in genomics, rational drug design and innate immune signaling can contribute to the design of nucleic acid-based drugs which can play a significant role in combating these emerging threats. Nucleic acid-based drugs (NABDs) are increasingly being recognized as promising antiviral therapeutic agents due to their versatile mechanisms of action, safety and efficacy profiles in preclinical studies, and amenable to address drug resistance arising from viral mutations. Specifically, the antiviral activity of siRNAs, antisense oligonucleotides and microRNAs is mediated by sequence-specific gene-silencing, and broad-spectrum innate and antiviral immune responses can be elicited by toll-like receptor agonists as such double-stranded RNA and CpG oligonucleotides. The purpose of this lecture is to highlight selective classes of NABDs and to discuss their mechanisms of action, preclinical safety and efficacy studies, current state of development, and provide future perspectives on the overall development of NABDs against emerging zoonotic viral diseases. In addition, challenges and barriers to the clinical development of NABDs and drug development against rare and neglected zoonotic viral diseases will also be highlighted in this review. In summary, NABDs are well suited to address emerging zoonotic viral diseases because they can be designed with great flexibility to match prevalent strains of zoonotic viruses, are robust and stable in many environmental conditions, and are versatile in mechanism of action to address viral mutations and drug resistance.

Nutraceutical Drug Discovery and Therapy (Track)

NEUTRACEUTICALS IN THE PREVENTION AND TREATMENT OF ATHEROSCLEROSIS

Thomas S. Davies, Hayley Gallagher, Joe W. E. Moss, Wijdan Al-Ahmadi and Dipak P. Ramji

Cardiff School of Biosciences, Cardiff University, Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK;

Atherosclerosis, the underlying cause of myocardial infarction and cerebrovascular accident, is responsible for most global deaths. Although there has been a slight recent reduction in mortality from this disease, at least in the western world, this is anticipated to change in the future due to worldwide increase in diabetes and obesity. Current therapies against atherosclerosis such as statins are associated with substantial residual risk together with issues of tolerability, side effects and patient-dependent efficacy. In addition, many pharmaceutical agents against other targets, such as inhibitors of cholesterol ester transfer protein and lipoprotein-associated phospholipase A2, have proved disappointing at the clinical level. There is therefore a need to develop alternative therapies, which could be taken in isolation or in conjunction with statins, for the prevention and treatment of this disease. Nutraceuticals represent promising alternatives but require an in-depth understanding of their actions together with the underlying mechanisms.

We have been investigating the impact of the immune and inflammatory responses via cytokines on atherosclerosis using in vitro and in vivo approaches for several years with focus on macrophages, which play pivotal roles in all stages of the disease. We have investigated the effects of several promising neutraceuticals, including hydroxytyrosol and dihomo-gamma-linolenic acid, on several key processes associated with atherosclerosis such as monocytic migration, macrophage polarization, foam cell formation, inflammasome activation and production of reactive oxygen species. These will be presented in the context of current therapies and future developments.

Pharmacogenomics  (Track)

CLINICAL UTILITY OF PHARMACOGENOMIC TESTING TO SUPPORT PRESCRIPTIVE DECISION MAKING IN PAIN MANAGEMENT

J. Dru Riddle

Texas Christian University, School of Nurse Anesthesia Fort, Worth, Texas 76108, USA

Introduction: Patient pharmacogenomic testing (PGX) was developed and later a version of it approved for use internationally to improve patient outcomes in response to prescribed medications. PGX testing uses genetic information to predict how a patient might respond to medications. Software is now available to interpret the testing results and support prescriptive decision-making in the clinical setting. However, uptake of this technology is slow. The study of clinical utility perceptions of pharmacogenomic testing to support clinical decision-making is not well reported. This study aims to describe facets of the multidimensional phenomena of clinical utility as defined in the ACCE model conceptual framework.

Methods: Mixed-method sequential qualitative-quantitative case study methodology design was used in this study. Framed by the ACCE model, a purposive sample of 12 anesthesia and pain management providers were interviewed using a focused interview guide framed by the ACCE framework regarding provider attitudes, beliefs, and knowledge regarding PGX technology. Multiple embedded case methodologies were used to analyze the results of the qualitative interviews. Findings from the qualitative data were used to construct a qualitative survey that was electronically distributed to 6,000 actively practicing Certified Registered Nurse Anesthetists in the United States. Results of the survey were analyzed using Horn’s parallel factor analysis to explain total variance and determine the number of factors that emerged.

Results: Seven themes emerged from the qualitative analysis: lack of understanding about PGX testing, lack of facilities to order and interpret PGX tests, concerns related to economic costs of PGX testing, PGX testing could be advantageous to patient outcomes, providers’ concerns for the ethical and legal implications of genetic testing, concern about the complexity of the testing and interpretation, and PGX testing could help avoid complications. Quantitative data revealed three distinct factors related to clinical utility of pharmacogenomic testing among anesthesia providers: Benefit (48% residual), knowledge (52% residual), and concerns (51% residual). Ninety seven percent of all factors loaded on these three findings.

Discussion/Conclusion: These findings indicate that anesthesia providers feel PGX testing could benefit patients and decrease complications; however, significant knowledge deficits and concerns related to complexity and ethical issues are currently prohibiting PGX testing from being used widely in anesthesia care. Interventions aimed at increasing provider knowledge about PGX testing are necessary to move PGX testing forward in clinical anesthesia care.

Keywords: Pharmacogenomic testing, clinical utility, anesthesia, factor analysis, mixed-method.

Pharmaceutical Biotechnology  (Track)

L-ASPARAGINASE 1 FROM SACCHAROMYCES CEREVISIAE AS NEW BIOPHARMACEUTICAL TO TREAT LEUKEMIA

Costa I.M, Leite M.M.S., Pedra, B.A.B., Schultz, L., Pessoa, A., Farsky, S.H.P., Oliveira, M.A. and Gisele Monteiro de Souza

Universidade de São Paulo – Faculdade de Ciências Farmacêuticas – Departamento de Tecnologia Bioquímico-Farmacêutica, Brazil

Leukemic cells depend on the presence of L-asparagine (Asn) in the bloodstream for protein synthesis, cell proliferation and survival. L-asparaginase (ASNase) is an important biopharmaceutical used in the treatment of the childhood leukemia, since this enzyme hydrolyzes Asn resulting in ammonia and aspartic acid. In consequence, tumor cells are prevented of using such amino acid for synthesis of essential proteins, leading to apoptotic cell death. ASNase is currently obtained from bacteria and formulations are associated with a high rate of adverse effects, mainly drug resistance and severe hypersensitivity, causing demand for alternative ASNase. The yeast Saccharomyces cerevisiae has the gene ASP1 responsible for the production of L-asparaginase 1 (ScASNase1) that possesses high similarity in amino acids sequence with the bacterial enzymes used as biopharmaceutical. This enzyme was previously characterized as non-promising antineoplastic agent, because the protein obtained endogenously from yeast presented a low specific activity and a low affinity to Asn. Because the bloodstream Asn concentration is up to 50 μM, an effective antineoplastic ASNase needs to present a high activity even in the presence of low substrate concentration. Thus, this work aims to verify the catalytic efficiency of heterologous produced ScASNase1, characterize the catalytic site and the kinetic parameters to compare them with bacterial enzyme ones, assessing whether ScASNase1 may represent an interesting therapeutic alternative in the treatment of Acute Lymphoblastic Leukemia. The ScASNase1 was isolated from the S. cerevisiae genomic DNA and cloned into NdeI and BamHI restriction sites of pET15b expression vector, which was cloned in E. coli (BL21(DE3)) for protein expression. The protein was purified by metal affinity chromatography. The ScASNase1 showed an specific activity of 200 U/mg, K0,5 = 75 μM with a Hill coefficient value of 2.005 indicating positive cooperativity of the enzyme with substrate. The catalytic efficiency was 3x106 M-1s-1 very similar to bacterial enzyme efficiency - 1 x106 M-1s-1. The reactions of site directed mutagenesis at residues of predicted catalytic site (T64A, T141A, K215A and Y78A) were performed and the proteins with mutations T64A, T141A, K215A and Y78A showed loss of 99% activity compared to wild type. The ScASNase1 was characterized in vitro in relation to cytotoxic effect in leukemia cells MOLT-4 and presented antitumor activity. Our results suggest that ScASNase1 shows allosteric feature and considering the catalytic efficiency and specific activity, it seems to be promising as biopharmaceutical.

Keywords: l-asparaginase, Saccharomyces cerevisiae, biopharmaceutical.

Pharmaceutical Research & Development  (Track)

DESIGN, DEVELOPMENT AND IN VITRO EVALUATION OF HYDROGELS AS CONTROLLED DRUG DELIVERY SYSTEM

Apurba Talukdar, B. K. Dey and Satish C.S.

Institute of Pharmacy, down town group of Institution, Assam down town University, Guwahati, India

In the present study Rosiglitazone was formulated in the form of hydrogels as a controlled drug delivery which enhances the therapeutic activities of the drug. The hydrogels were synthesized by free radical polymerization technique using N,N′-Methylene bisacrylamide as crosslinker and potassium persulphate as polymerization initiator to polymerize acrylic acid (AAc) to poly(acrylic acid) in presence of polyvinylpyrrolidone (PVP) to form a polymeric network. The concentration of polymers and crosslinking agents were varied in order to study the swelling, equilibrium swelling ratio (ESR), diffusion coefficients and in-vitro drug release profiles. The drug polymers compatibility was confirmed by Fourier transform infrared spectroscopy (FT-IR) and Differential scanning calorimetry (DSC). The X-ray diffraction (XRD) studies were carried out to check the nature of the drug in the formulations. Surface morphology was studied by using Scanning electron microscopy (SEM). Equilibrium swelling ratio showed that the swelling was pH depended and at pKa value (3-4) of acrylic acid the swelling showed a decreased value. From the diffusion study it was concluded that as the acrylic acid concentration increased and crosslinker decreased the diffusion coefficient was increased. The in-vitro release data showed that, as the concentration of acrylic acid was increased; swelling increased resulting in increased release of the drug. But the drug release was found to get decreased when the concentration of PVP and crosslinker was increased. From the results, it can be deduced that the AAc-PVP hydrogel could be suitable as a controlled drug delivery system for the antidiabetic drug Rosiglitazone.

Keywords: Rosiglitazone, Hydrogels, acrylic acid, polyvinylpyrrolidone.

Pulmonary Drug Discovery and Therapy  (Track)

POTENTIAL THERAPEUTIC TARGETS FOR TREATING CORTICOSTEROID INSENSITIVITY IN SEVERE ASTHMA

Yassine Amrani

Institute for Lung Health, Department of Respiratory Medicine, Clinical Sciences Building, Glenfield Hospital, Groby Rd, Leicester, LE3 9QP, U.K.

An approximate of 5 to 10% of patients have difficult-to-control or severe disease that is poorly responsive to glucocorticoid (GC) therapy. The management of these severe patients with asthma requires the chronic administration of high doses of GC (oral or high-dose inhaled GC) which is often associated with serious adverse effects. In addition, severe asthma represents a significant unmet clinical need since these patients are responsible for more than 50% of the total asthma-related health care costs and morbidity. Therefore, better anti-inflammatory therapies are urgently needed to properly manage GC resistance in asthma.

Growing in vivo evidence supports the concept that airway smooth muscle (ASM) produces various immuno-modulatory factors that could contribute to severe asthma pathogenesis via the regulation of airway inflammation, airway narrowing and remodelling. Targeting ASM using bronchial thermoplasty has provided undeniable clinical benefits for patients with uncontrolled severe asthma who are refractory to glucocorticoid therapy. The purpose of this presentation will support the view that the failure of glucocorticoids to adequately manage patients with severe asthma could derive from their inability to affect the immuno-modulatory potential of ASM. We will provide evidence that ASM sensitivity to glucocorticoid therapy can be blunted in severe asthma and will describe some of the mechanisms that could represent potential targets for the treatment of glucocorticoid insensitivity.

Pulmonary drug discovery and Therapy  (Track)

EXTRA FINE PARTICLE INHALED CORTICOSTEROIDS, PHARMACOKINETICS AND SYSTEMIC ACTIVITY IN CHILDREN WITH ASTHMA

Ole D Wolthers

Asthma and Allergy Clinic, Children´s Clinic Randers, Clinical Institute, Health, Aarhus University, Denmark

During recent years extra fine particle inhaled corticosteroids with a median aerodynamic diameter ≤ 2 μm have been introduced in the treatment of asthma. The aim of the present study was to review pharmacokinetics and systemic activity of extra fine particle hydroalkane pressurised metered dose inhaled (pMDI) corticosteroids in children. Ciclesonide and beclomethasone dipropionate pMID are the most widely available extra fine particle formulations. The rate of pulmonary deposition varies from < 10% in conventional chloroflourocarbon pMDIs to 51 and 52% of the delivered dose in extra fine ciclesonide and beclomethasone dipropionate pMDIs. Systemic bioavailability of oral and pulmonary deposition is 52 and 82% of extra fine ciclesonide and beclomethasone dipropionate, respectively. Secondary analyses of measures of hypothalamicpituitary- adrenal function found no effects of ciclesonide pMDI between 40 and 320 μg/day or of beclomethasone dipropionate pMDI between 80 and 400 μg/day. Ciclesonide pMDI 40, 80 and 160 μg/day caused no effects on short term lower leg growth rate as assessed by knemometry, whereas 320 μg/day was associated with a numerically suppression equivalent to 30%. Systemic activity was evidenced by a number of randomized, controlled knemometry studies of extra fine beclomethasone dipropionate pMDI; 200 μg/day was non-inferior to conventional chlorofluorocarbon beclomethasone diproponate pMDI 200 and 400 μg/day. In conclusion, key pharmacokinetic features of pMDIs are altered by extra fine formulations of inhaled corticosteroids explaining the evidence of systemic activity as detected by knemometry. Implications for systemic activity of doses of extra fine beclomethasone dipropionate < 200 μg/day and of ciclesonide >180 μg/day remain to be further studied.

Regenerative Medicine  (Track)

CARDIAC ATRIAL APPENDAGE STEM CELLS ENGRAFT AND DIFFERENTIATE INTO CARDIOMYOCYTES IN VIVO: A NEW TOOL FOR CARDIAC REPAIR AFTER MI

Yanick Fanton, Annick Daniëls, Boris Robic, Jean-Luc Rummens, Severina Windmolders, Leen Willems, Luc Jamaer, Jasperina Dubois, Eric Bijnens, Nic Heuts, Kristof Notelaers, Rik Paesen, Marcel Ameloot, Urbain Mees, Virginie Bito, Jeroen Declercq, Karen Hensen, Remco Koninckx and Marc Hendrikx

Faculty of Medicine and Life Sciences, Hasselt University, Hasselt, Belgium

Recently, a new cardiac stem cell population was identified, called the cardiac atrial appendage stem cell (CASC). The safety and efficacy of autologous CASC transplantation in a minipig MI model was examined here together with assessment of possible tumorigenic potential for CASCs.

CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and labeled with green fluorescent protein (GFP). After cell expansion, MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were trans-endocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. Left ventricular (LV) function was assessed by 3D electromechanical mapping (EMM) and cardiac MRI.

LV dilatation was observed in MI pigs at two months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16±8% in MI pigs vs 3±10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC.

qPCR against the GFP gene revealed a high CASC cell retention of 19%. This was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, shown by a sarcomeric expression of cardiac troponin T and I. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in CASC-derived cardiomyocytes. Moreover, functional integration was demonstrated by expression of the gap junction protein Cx43 between CASCs aligned with surviving cardiomyocytes, which was confirmed by the absence of cardiac arrhythmias.

Possible tumorigenic properties of CASCs, isolated from human atrial appendages, were explored in immunocompromised mice. One million CASCs dissolved in Matrigel were injected in Foxn1nu mice. After 6 months none of the mice developed subcutaneous tumors, while all mice injected with a UM-SCC tumor cell line had to be sacrificed with large subcutaneous tumors after 2-3 weeks (n=10 for each group). This demonstrates that CASCS do not show any tumorigenic potential.

In conclusion, CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Furthermore, this study provides a first proof of the safety of CASC therapy. CASCs thus seem very promising for myocardial repair after MI.

Stereoselective Synthesis of Bioactive Compounds (Track)

PHARMACEUTICALLY FAVORED HETEROCYCLES VIA BAYLIS-HILLMAN CHEMISTRY

V.Jayathirtha Rao

Crop Protection Chemicals Division, AcSIR-IICT, csir-Indian Institute of Chemical Technology, Uppal Road Tarnaka, Hyderabad – 500007, India

Heterocycles form by far the largest of the classical divisions of organic chemistry and are of immense importance biologically, industrially, and indeed to the functioning of any developed human society. Many heterocyclic compounds are biosynthesized by plants and animals and are biologically active. The biological properties of heterocycles in general make them one of the prime interests of the pharmaceutical industry and biotechnology industry. In this context we have initiated work on the synthesis of heterocycles using Baylis-Hillman reaction. The prominent features of BH reaction, like simple reaction conditions, readily available and cheap chemicals, atom economy, ability to produce densely functionalized products, control of stereochemistry and selectivity are the driving or attractive points for medicinal chemists to utilize and exploit in making variety of chemicals having pharmaceutical importance. The objective/aim of the presentation is to comprehend our results on medicinally important publications in connection with BH chemistry and further provide guidelines to use this reaction for the synthesis of pharmaceutically important entities. The work presented on this subject will definitely have an impact over pharmaceutical companies.

Women’s Health Drug Discovery & Therapy (Track)

EFFECT OF PENEHYCLIDINE HYDROCHLORIDE ON CALCIUM CONCENTRATION AND M RECEPTOR DENSITY OF RAT UTERUS CELL

Hong-Tao Xiao^1,2 and Rong-Sheng Tong^1,2

¹School of Medicine, University of Electronic Science and Technology of China, Chengdu, China;
²Department of Pharmacy, Hospital of the University of Electronic Science and Technology of China and Sichuan Provincial People's Hospital, Chengdu, China; Address: NO. 32, West Second Section, First Ring Road, Chengdu, Scihuan China;

We investigated the effect of penehyclidine hydrochloride(PHC), a novel selective anticholinergic drug on calcium concentration and M receptor density of rat uterus cell. The methods of Fura-2/AM and radioimmunity assaies were further employed to determine calcium concentration and M receptor density levels in rat uterus cell for comparing with uterus contraction mechanisms of PHC. In the assaies, PH could reduce the calcium concentration and the M receptor density of the rat uterus smooth muscle and at high dose, medium dose and low dose. In conclusion, our results suggested that PHC has potential as a candidate drug for women suffering from dysmenorrheal and other sexual pain disorders, by acting on calcium channel and the M receptor.

Keywords:Penehyclidine hydrochloride, calcium concentration, M receptor density.