Issue 3 2007

RNAi: an attractive choice for future therapeutics

Issue 3 2007 / 23 May 2007 / John J. Rossi, Division of Molecular Biology, Beckman Research Institute of the City of Hope, Graduate School of Biological Sciences, Duarte, United States

RNA interference (RNAi) is a regulatory mechanism of most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules as triggers to direct homology-dependent control of gene activity (Almeida and Allshire 2005).

Known as small interfering RNAs (siRNA) these ~21-22 bp long dsRNA molecules have characteristic 2 nucleotide 3’ overhangs that allows them to be recognised by the enzymatic machinery of RNAi that eventually leads to homology-dependent degradation of the target mRNA. In mammalian cells siRNAs are produced from cleavage of longer dsRNA precursors by the RNaseIII endonuclease Dicer (Zhang et al. 2004). Dicer is complexed with two RNA binding proteins; the TAR-RNA binding protein (TRBP) and PACT, which are involved in the hand off of siRNAs to the RNA-induced silencing complex (RISC)(Lee et al. 2006). The core components of RISC are the Argonaute (Ago) family members, In humans there are eight members of this family but only Ago-2 possesses an active catalytic domain for cleavage activity (Liu et al. 2004; Meister et al. 2004). While siRNAs loaded into RISC are double-stranded, Ago-2 cleaves and releases the “passenger” strand leading to an activated form of RISC with a single-stranded “guide” RNA molecule that directs the specificity of the target recognition by intermolecular base pairing (Tang 2005). Rules that govern selectivity of strand loading into RISC are based upon differential thermodynamic stabilities of the ends of the siRNAs (Khvorova et al. 2003; Schwarz et al. 2003). The less thermodynamically stable end is favoured for binding to the PIWI domain of Ago-2. (more…)

Tagged with: Beckman Research Institute of the City of Hope, John J. Rossi, RNAi, siRNA Therapeutics

Biomarker discovery and validation in clinical proteomics

Issue 3 2007 / 23 May 2007 / Professor Stephen R Pennington, Proteome Research Centre, Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin

Until recently the use of proteomics in the biomedical arena has included programmes aimed at the elucidation of cellular responses to extracellular stimuli by known and potential drugs. It has been anticipated that these will lead to the elucidation of the basic mechanisms of cellular responses, potential identification of new drug targets and discovery of the mechanism of action of drugs both NCE’s and those currently in development.

In addition, the use of proteomics to investigate toxic responses to drugs (toxicoproteomics) in pre-clinical and clinical studies has gained increasing importance, particularly when such data is analysed in the context of more traditional toxic responses. Notably, many of these studies are now undertaken in the context of multi-team groups or a consortia of researchers from different disciplines – including proteomics. An interesting example of such a consortium is the EU and commercially funded ‘PredTox’ predictive toxicology programme being undertaken by a range of companies and Universities; the programme forms part of the ‘Innovative Medicines for Europe’ initiative. There are important lessons to be learnt from the operational management of such consortia (to be discussed further on within the article). The PredTox programme is exemplary of the growing interest in the use of ‘omics technologies, including proteomics, to identify ‘biomarkers’, as well as a vibrant debate on the relative importance of biomarkers for revenue generation in the pharma sector. Here, the term ‘biomarkers’ will be defined in the context of proteomics, an historical overview of the evolution of proteomics technologies and their continuing development will be given, and some recent biomarker discovery projects will be outlined with the use of selected examples. To conclude, some of the lessons learnt and challenges that remain in biomarker discovery, validation and implementation will be summarised. (more…)

Tagged with: Biomarkers, Conway Institute of Biomolecular and Biomedical Sciences, Professor Stephen R Pennington, Proteomics, University College Dublin

The handling and analysis of large scale high content screening data

Issue 3 2007 / 23 May 2007 / Karol Kozak and Benjamin Eshun, Data Handling in TDS, Max Planck Institute of MolecularCell Biology and Genetics, Germany. Jeff Oegema, CEO, Scionics Computer Innovation, GmbH

Data management has become one of the central issues in High Content Screening (HCS) as it has high potential within predictive toxicity assessments. In particular, HCS applying automated microscopy requires a technology and system which is capable of storing and analying vast amounts of image and numeric data. HCS data includes comprehensive information about the bioactive molecules, the targeted genes and images, as well as their extracted data matrices after acquisition. Here we describe a bioinformatics solution HCS LIMS (Laboratory Information Management System) for the management of data from different screening microscopes. Additionally, the data handling approaches used in HCS for image converting, compression and archiving of images are discussed. (more…)

Tagged with: Benjamin Eshun, HCS (High Content Screening), Jeff Oegema, Karol Kozak, Laboratory information management systems (LIMS), Max Planck Institute, Scionics Computer Innovation GmbH

Launching an HTS campaign to discover new antivirals

Issue 3 2007 / 23 May 2007 / Colleen B. Jonsson, Ph.D., Program Leader, Emerging Infectious Disease Research and E. Lucile White, Manager, High-Throughput Screening Center and Enzymology Laboratory, Southern Research Institute, Birmingham, AL, United States

There are over 300 human viruses that have no treatment, vaccine or antiviral. Unfortunately, only sixty-two drugs are approved by the US Food and Drug Administration (FDA) for the treatment of six different viral illnesses. Of these, 45% are for the treatment of HIV/AIDS. The remaining drugs offer treatments that target hepatitis B and C, herpes, influenza, and respiratory syncytial viruses. Antiviral drugs can play a significant role in the containment of an outbreak of an emerging virus. Vaccination of individuals during an outbreak can also prove effective; however, protection of an individual from the threat may not occur for two or more weeks after the initial vaccination. Hence, only a drug can be offered as a prophylactic treatment of individuals in an endemic area.

The following case study presents the Southern Research Institute (SRI) approach to enabling the rapid discovery of new antiviral lead compounds for the treatment of the influenza virus1, which has also been successfully applied to SARS CoV2 and other emerging viruses. High throughput screening is a system used in pharmaceutical and biotechnology companies to screen a large number of compounds in a short period of time. In screening a library of over one million compounds, we have demonstrated that three primary areas should be taken into consideration: (1) adapting cell-based assays for HTS (2) logistics of screening and assessing the results from 1,000,000 compounds, and (3) confirming hits and obtaining early mechanism of action information. (more…)

Tagged with: Antivirals, Colleen B. Jonsson Ph.D., E. Lucile White, HTS (High Throughput Screening), Southern Research Institute

Calorimetry for amorphous content quantification

Issue 3 2007 / 23 May 2007 / Simon Gaisford PhD and Rita Ramos PhD, School of Pharmacy, University of London

In the previous article (European Pharmaceutical Review, Issue 2, 2007) an introduction to calorimetry was given and its application to polymorph characterisation, discussed. Another area of application of growing importance is quantification of (usually small) amorphous contents. A requirement to demonstrate the presence or absence of amorphous material is becoming more important in regulatory documentation and calorimetric techniques are emerging as major tools in this arena. This article focuses on the use of various calorimetric techniques for quantifying amorphous content. (more…)

Tagged with: Amorphous Content Quantification, Calorimetry, Rita Ramos PhD, Simon Gaisford PhD, Thermal Analysis, University of London

Ion channel electrophysiology in pharmaceutical research

Issue 3 2007 / 23 May 2007 / Daniel Konrad, Simon Hebeisen, and Urs Thomet, bSys GmbH

Ion channels are well recognised as targets for a vast range of disease states and conditions. The process of discovering drugs is influenced by the biological confidence in the rationale of the screening approach and the screenability. Various methods have been gathered around the gold standard of manual patch-clamping that account for the higher number of datapoints in functional electrophysiology.

Real UHTS, but non-functional fluorescence or ligand binding methods, were recently added by various automated patch clamp techniques that could partially establish datapoints in a midthroughput range. Since the establishment of reliable high quality data remains crucial in drug discovery and safety assessment, the chronology of screening technologies applied in the discovery remains important. This article provides background information on ion channel electrophysiology, as well as an overview of its current employment in pharmaceutical discovery and drug development.

Ion channels are membrane-bound proteins that allow the passage of ions across impermeable cell membranes. Their most obvious role is to generate action potentials for cardiac and neuronal cells. In addition, they have other extensive functions in cell volume control, differentiation and growth of cells and neurotransmitter release. Structurally, they can be divided into two major families; voltage-gated and ligand-gated ion channels. (more…)

Tagged with: bSys GmbH, Daniel Konrad, Ion channel electrophysiology, Simon Hebeisen, Urs Thomet

Automated classification of crystallisation images

Issue 3 2007 / 23 May 2007 / Julie Wilson, York Structural Biology Laboratory, Department of Chemistry, University of Heslington, York, UK

The findings of many crystallisation experiments are required in order to identify conditions that will produce diffraction quality crystals. The use of robots has increased the number of experiments performed in most laboratories and, in structural genomics centres, tens of thousands of experiments can be produced every day. As each experiment must be assessed regularly over a period of time, visual inspection is becoming increasingly impractical and automated imaging systems are now available to record the results; the aim of this research is the development of software to analyse and classify images from crystallisation trials.

Structural genomics aims to implement high-throughput technologies in order to speed up molecular structure determination; however, the process of obtaining diffraction quality crystals continues to present a major bottleneck. Currently there is no a priori method to determine the optimum crystallisation strategy for a particular protein, and the process remains highly empirical. Several important variables, which often interact; such as protein concentration, precipitant, pH and temperature must be tested in combination. Although various sampling techniques have been proposed to reduce the number of possible combinations, the screening of conditions is a largely trial-and-error process. (more…)

Tagged with: Julie Wilson, Protein crystallography, University of Heslington

Spray-freeze-drying in the manufacture of pharmaceuticals

Issue 3 2007 / 23 May 2007 / Heiko A. Schiffter, Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford

Over the last decade, the development of new drug delivery methods and devices for dry powder inhalation1, needle-free intradermal powder injection2 or sustained parenteral drug delivery3 has led to an increasing demand for powder formulations incorporating an active pharmaceutical ingredient (API)4,5. (more…)

Tagged with: Freeze Drying, Heiko A. Schiffter, Manufacturing, University of Oxford

PAT and design science

Issue 3 2007 / 23 May 2007 / Carl-Fredrik Mandenius, Micael Derelöv, Jonas Detterfelt, Mats Björkman, Division of Biotechnology/IFM and Division of Production Systems/IKP, Linköping University, Sweden

Process analytical technology (PAT) and mechanical design science are interconnected; this article describes how a well-established design modelling approach; the Hubka-Eder model, is applied to the concepts of PAT and quality by design (QBD). The model connects PAT with quality management concepts as defined for PAT by the ICH guidelines for quality issues. Examples are taken from biopharmaceutical applications, but these are also applicable to other active pharmaceutical ingredients (API). Benefits of using a conceptual design modelling approach in PAT and related subjects are discussed and suggested as a complementary functionality analysis tool in PAT and quality design of pharmaceutical processes. (more…)

Tagged with: Carl-Fredrik Mandenius, Design science, Jonas Detterfelt, Linköping University, Mats Björkman, Micael Derelöv, PAT

EuPAT: an initiative to promote progress in the science underpinning PAT

Issue 3 2007 / 23 May 2007 / Staffan Folestad, Senior Principal Scientist, AstraZeneca, Sweden; Peter York, Bradford University, UK, and Rasmus Bro, Copenhagen University, Denmark

A new initiative launch has been announced that aims to promote progress in the science underpinning Process Analytical Technology. The core purpose of the EuPAT meeting is the creation of an open and neutral scientific forum for sharing and discussing new findings in cutting-edge scientific research, development of enabling technologies and development of innovative applications. Building on the successful EuPAT1 conference in Gothenburg, November 2006; the EuPAT2 conference has been announced for November 2007 in Copenhagen. (more…)

Tagged with: AstraZeneca, Bradford University, Copenhagen University, EuPAT, PAT, Peter York, Rasmus Bro, Staffan Folestad

The significance and detection of VBNC microorganisms

Issue 3 2007 / 23 May 2007 / Dr Paul Newby, Team Leader, Pharmaceutical Microbiology, GlaxoSmithKline

The purpose of this article is to assess the potential significance of Viable but Non Culturable (VBNC) microorganisms in the pharmaceutical industry; consideration is given to the definition of the VBNC state, current methods for the detection of such organisms are outlined and potentially significant new methods, which may impact the industry, are considered. The article will also discuss the future significance of VBNC organisms within pharmaceutical microbiology and its ongoing development. (more…)

Tagged with: Dr Paul Newby, GlaxoSmithKline, Viable but Non Culturable (VBNC) microorganisms