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PCR Viability
PCR Viability

PCR and personalised cancer medicine

16 December 2010 | By Frank McCaughan, MRC Career Development Fellow, MRC Laboratory of Molecular Biology

The delivery of personalised medicine is a key goal of modern cancer medicine and refers to the tailoring of anticancer therapy to the molecular characteristics of an individual tumour. To facilitate personalised medicine, it is important to have robust and reproducible means of gaining molecular information about a patient’s cancer…

Figure 1 Schematic illustration of arrayed or pooled RNAi screens in cells. Left panel. Pooled-viral vectors encoding libraries of shRNAs targeting multiple genes can be used to transduce a target cell population in a single tissue culture dish. After selection for the desired phenotype, cells are analysed for the identification of genes whose inhibition by RNAi knockdown cause the specific phenotype as described in Table 1. The relative abundance of each shRNA or a random 60-mer barcode expressed from the same vector as the specific shRNA can be identified and quantified by labelling the PCR product with fluorescent dyes (e.g., Cy5 or Cy3). The PCR products are then hybridised to custom designed cDNA microarrays containing barcode or shRNA complementary oligonucleotides. The relative abundance of barcodes obtained from the cells that were exposed to selective pressure are compared to that detected in control cells that have been exposed to the same shRNA library, but not to the selective pressure (for example, drug treatment or genetic mutations). Right panel. Arrayed RNAi screen libraries consist of individual siRNA or shRNA reagents that target different genes and that are placed in each well of a multi-well plate. RNAi reagent libraries can comprise synthetic siRNAs, plasmid-or virally-encoded shRNAs. Various assay readouts are used to determine the effect of RNAi on the phenotype as described in Table 1. Adapted10.
Figure 1 Schematic illustration of arrayed or pooled RNAi screens in cells. Left panel. Pooled-viral vectors encoding libraries of shRNAs targeting multiple genes can be used to transduce a target cell population in a single tissue culture dish. After selection for the desired phenotype, cells are analysed for the identification of genes whose inhibition by RNAi knockdown cause the specific phenotype as described in Table 1. The relative abundance of each shRNA or a random 60-mer barcode expressed from the same vector as the specific shRNA can be identified and quantified by labelling the PCR product with fluorescent dyes (e.g., Cy5 or Cy3). The PCR products are then hybridised to custom designed cDNA microarrays containing barcode or shRNA complementary oligonucleotides. The relative abundance of barcodes obtained from the cells that were exposed to selective pressure are compared to that detected in control cells that have been exposed to the same shRNA library, but not to the selective pressure (for example, drug treatment or genetic mutations). Right panel. Arrayed RNAi screen libraries consist of individual siRNA or shRNA reagents that target different genes and that are placed in each well of a multi-well plate. RNAi reagent libraries can comprise synthetic siRNAs, plasmid-or virally-encoded shRNAs. Various assay readouts are used to determine the effect of RNAi on the phenotype as described in Table 1. Adapted10.

RNAi screens for the identification and validation of novel targets: Current status and challenges

16 December 2010 | By Attila A. Seyhan, Translational Immunology, Inflammation and Immunology, Pfizer Pharmaceuticals

Recent advances in RNA interference (RNAi) technology and availability of RNAi libraries in various formats and genome coverage have impacted the direction and speed of drug target discovery and validation efforts. After the introduction of RNAi inducing reaagent libraries in various formats, systematic functional genome screens have been performed to…

The implementation of rapid microbiological methods

16 December 2010 | By

This is the sixth and final paper in a series of articles on rapid microbiological methods that have appeared in European Pharmaceutical Review during 2010. Over the past year, we have explored the world of rapid microbiological methods (RMMs), focusing on validation strategies, regulatory expectations, and the technical and quality…

PharmaChemical Ireland launch innovation and excellence strategy in New York

16 December 2010 | By Matt Moran, Director, PharmaChemical Ireland

PharmaChemical Ireland published a major strategy document in March 2010. The document, titled, ‘Innovation and Excellence-PharmaChemical Ireland Strategic Plan’ was launched at the Drug Chemical and Allied Technologies (DCAT) meeting at the Waldorf Astoria, New York City on 16 March 2010. The document outlines the industry response to the major…

Figure 1 A: Control B: Docetaxel treated Induction of apoptosis with Docetaxel compared to untreated. The lower right quadrant represents cells in early apoptosis and cells in the upper right quadrant are cells in late apoptosis due to PI entering the cells through leaky membranes. C: Control D: Docetaxel treated Cell cycle of control cells and Docetaxel treated cells. The first peak shows cells in G0/G1 and the second peak is cells in G2/M. The breast cancer cell line, MDA MB231, was treated with 10nM docetaxel or DMSO control for 72 hours. Cells were harvested, the nuclei isolated and stained with PI. Samples were run on the LSR II flow cytometer and analyed in FCS Express software. Docetaxel arrests the cells in G2/M
Figure 1 A: Control B: Docetaxel treated Induction of apoptosis with Docetaxel compared to untreated. The lower right quadrant represents cells in early apoptosis and cells in the upper right quadrant are cells in late apoptosis due to PI entering the cells through leaky membranes. C: Control D: Docetaxel treated Cell cycle of control cells and Docetaxel treated cells. The first peak shows cells in G0/G1 and the second peak is cells in G2/M. The breast cancer cell line, MDA MB231, was treated with 10nM docetaxel or DMSO control for 72 hours. Cells were harvested, the nuclei isolated and stained with PI. Samples were run on the LSR II flow cytometer and analyed in FCS Express software. Docetaxel arrests the cells in G2/M

Application of flow cytometry in drug discovery

16 December 2010 | By Dana Buckman, Senior Scientist, Biomarkers – Translational Research, Pfizer

Flow cytometry can be used to advance our understanding of diseases in multiple ways. Drug effects and dosages can be ascertained in vitro, along with patient selection based on mutations and antigen profiles. Within the Diagnostic Biomarkers group of Translational Research at Pfizer, we are utilising flow cytometry in conjunction…

Figure 1 Schematic illustration of the recombinant antibody microarray set-up
Figure 1 Schematic illustration of the recombinant antibody microarray set-up

Developing and applying recombinant antibody microarrays for high-throughput disease proteomics

16 December 2010 | By Carl A.K. Borrebaeck and Christer Wingren, Department of Immunotechnology and CREATE Health, Lund University

Deciphering crude proteomes in the quest for candidate biomarker signatures for disease diagnostics, prognostics and classifications has proven to be challenging using conventional proteomic technologies. In this context, affinity protein microarrays, and in particular recombinant antibody microarrays, have recently been established as a promising approach within high-throughput (disease) proteomics1-3. The…

Vaccines and needle
Vaccines and needle

Development of stabilised vaccines with needle-free devices for targeted skin immunisation

16 December 2010 | By Abina M. Crean & Anne C. Moore, School of Pharmacy, University College Cork and Conor O’Mahony, Tyndall National Institute, University College Cork

Vaccination represents the primary public health measure to combat infectious diseases. However, limitations of cold-chain storage, vaccine wastage, hazardous sharps-waste and the requirements for trained personnel add significant and unsustainable financial and logistic costs to immunisation programmes. Developments of needle-free methods should aim to overcome these logistics issues from the…

Figure 1 Key signaling pathways of Epidermal Growth Factor Receptor (EGFR). The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) superfamily of receptors comprising of four distinct however structurally similar tyrosine kinase receptors. Upon ligand binding (e.g. EGF, TGF-α) EGFR dimerises with another receptor and undergoes phosphorylation of its TK domain. Activated EGFR stimulates cell proliferation, survival, migration, adhesion and differentiation. EGFR is associated with increased or inappropriate signaling in NSCLC and is a key mediator of tumor progression. Activating mutations of the EGFR kinase domain result in ligand-independent activation of the pathway. Tyrosine kinase inhibitors, such as erlotinib and gefitinib, interfere with the kinase activity of the gene and prevent downstream signaling. Therefore, EGFR is an important target for NSCLC treatment. Modified from Gazdar et al22
Figure 1 Key signaling pathways of Epidermal Growth Factor Receptor (EGFR). The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) superfamily of receptors comprising of four distinct however structurally similar tyrosine kinase receptors. Upon ligand binding (e.g. EGF, TGF-α) EGFR dimerises with another receptor and undergoes phosphorylation of its TK domain. Activated EGFR stimulates cell proliferation, survival, migration, adhesion and differentiation. EGFR is associated with increased or inappropriate signaling in NSCLC and is a key mediator of tumor progression. Activating mutations of the EGFR kinase domain result in ligand-independent activation of the pathway. Tyrosine kinase inhibitors, such as erlotinib and gefitinib, interfere with the kinase activity of the gene and prevent downstream signaling. Therefore, EGFR is an important target for NSCLC treatment. Modified from Gazdar et al22

Targeted therapies in lung cancer and Biomarkers

16 December 2010 | By Wolfgang M. Brueckl & Joachim Ficker, Department of Internal Medicine 3, Lung Cancer Center and Thomas M. Mundel, Roche Parma AG

Despite innumerable clinical studies in the past three decades with lots of traditional chemotherapeutical drugs and drug combinations, survival in lung cancer has increased by far less than other entities. Research now focuses on inhibitors of tyrosine kinases which have been shown to have a central role in the development…

Article 5: The implementation of rapid microbiological methods

1 November 2010 | By Michael J. Miller, President, Microbiology Consultants, LLC

This is the fifth in a series of articles on rapid microbiological methods that will appear in European Pharmaceutical Review during 2010. In my previous four articles, I have provided an overview of the benefits of rapid microbiological methods (RMMs) as compared with conventional methods, validation strategies and regulatory perspectives…

Figure 2 Spray drying. Screenshot of on-line PSD analysis, providing real-time PSD-measurement (top-right) and continuous PSD-monitoring (bottom; d50-orange, d90-blue, d10-green, transmission-red)
Figure 2 Spray drying. Screenshot of on-line PSD analysis, providing real-time PSD-measurement (top-right) and continuous PSD-monitoring (bottom; d50-orange, d90-blue, d10-green, transmission-red)

Applying PAT in pharmaceutical processes

1 November 2010 | By Mario Hellings, Tom Van den Kerkhof, Jeroen Geens and Steve Mehrman, Johnson & Johnson

As cited by the FDA, “Process Analytical Technology (PAT) is a system for designing, analysing, and controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality.”1 The main goal of PAT…

Figure 3 A multi-omics approach for the discovery and validation of biomarkers to probe multidimensional phases of disease biology. A robust biomarker discovery, development and validation effort must bring together multiple ‘omics’ technologies, data types, databases and bioinformatics and biostatics to identify the most predictive biomarkers across DNA, RNA, protein, phenotype and metabolite domains
Figure 3 A multi-omics approach for the discovery and validation of biomarkers to probe multidimensional phases of disease biology. A robust biomarker discovery, development and validation effort must bring together multiple ‘omics’ technologies, data types, databases and bioinformatics and biostatics to identify the most predictive biomarkers across DNA, RNA, protein, phenotype and metabolite domains

Biomarkers in drug discovery and development

1 November 2010 | By Attila A. Seyhan.Translational Immunology, Inflammation and Immunology, Pfizer

Robust and validated biomarkers are needed to improve diagnosis, monitor drug activity and therapeutic response and guide the development of safer and targeted therapies for various chronic diseases. While different types of biomarkers have been impactful in the field of drug discovery and development, the process of identifying and validating…

Figure 1 Principle of 2D cell migration assays. (A) A confluent monolayer of cells is mechanically wounded (‘scratch assay’), usually with a sterile pipette tip, leaving two wound edges (dashed lines) separated by a void. Cells at the leading edges quickly assume a polarised morphology and form broad lamellae pointing into the direction of the void. Over time, cells migrate into the void and, eventually, completely close the wound (B). Cell migration is qualitatively assessed by visual inspection and can be quantitated by measuring gap width or by enumeration of cells populating the wound. Images show T98G human glioblastoma cells immediately after wounding (A) or after 24 hours of migration (B). Distance between lines is one millimetre
Figure 1 Principle of 2D cell migration assays. (A) A confluent monolayer of cells is mechanically wounded (‘scratch assay’), usually with a sterile pipette tip, leaving two wound edges (dashed lines) separated by a void. Cells at the leading edges quickly assume a polarised morphology and form broad lamellae pointing into the direction of the void. Over time, cells migrate into the void and, eventually, completely close the wound (B). Cell migration is qualitatively assessed by visual inspection and can be quantitated by measuring gap width or by enumeration of cells populating the wound. Images show T98G human glioblastoma cells immediately after wounding (A) or after 24 hours of migration (B). Distance between lines is one millimetre

Advances in two-dimensional cell migration assay technologies

1 November 2010 | By Andreas Vogt, Department of Pharmacology and Chemical Biology and the University of Pittsburgh Drug Discovery Institute, University of Pittsburgh

Cell motility plays an important role in many human diseases and normal cellular processes. Cell migration is critical for wound healing as cells of the inflammatory system and fibroblasts populate the wound and initiate re-epithelialisation1. On the other hand, unregulated cell migration contributes to cancer cell invasion and metastasis2. Agents…

Functional genomics as a tool for guiding personalised cancer treatment

29 October 2010 | By Roderick Beijersbergen, Group Leader Molecular Carcinogenesis, the Netherlands Cancer Institute

Improved understanding of the molecular alterations in cancer cells has fuelled the development of more specific and directed cancer therapies. However, it has become clear that response rates can be low due to confounding genetic alterations that render these highly specific therapies ineffective. As a result, the costs of cancer…

Figure 1 siRNA drug discovery pipeline
Figure 1 siRNA drug discovery pipeline

The evolution of RNAi technologies in the drug discovery business

29 October 2010 | By Jason Borawski and L. Alex Gaither, Novartis Institutes for Biomedical Research

In the past decade, the pharmaceutical industry has exploited the naturally occurring cellular RNAi pathway to enhance drug discovery research. The RNAi pathway, triggered by dsRNA, selectively, although not always specifically, degrades mRNA leading to substantial decreases in post-transcriptional gene expression1. Researchers have capitalised on this intrinsic pathway by synthesising…

Figure 1 Distribution of drugs in vivo depends on various equilibrium and rate processes between compartments
Figure 1 Distribution of drugs in vivo depends on various equilibrium and rate processes between compartments

Bio-mimetic chromatography to predict drug distribution in vivo

29 October 2010 | By Klara Valko, Analytical Chemistry, GlaxoSmithKline Medicines Research Centre

A major concern for the pharmaceutical industry is the high attrition rate (>90 per cent) of potential drug molecules failing during late stages of the drug discovery process. This may be due to lack of efficacy in the clinic, unexpected side effects or unfavourable pharmacokinetics. There is a need for…