Recent advances in topical ophthalmic drug delivery with lipid-based nanocarriers

Ocular barriers and the poor water solubility of drug candidates present a number of problems for the development of ocular drug delivery systems. Recently, the emergence of lipid-based nanocarriers has provided a viable means of enhancing the bioavailability of ophthalmic formulations. A number of these formulations have been found to be clinically active and several others are currently undergoing clinical trials. In this review, the advantages of lipid-based nanocarriers as non-invasive topical ocular drug delivery systems are presented. Many systems, including emulsions, liposomes, cubosomes, niosomes and other lipid-based nanocarriers, are reviewed.

Particulate delivery systems for vaccines

This review focuses on the use of particulate delivery systems for the purposes of immunization. This includes poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, chitosan, and other biodegradable polymers. These systems are evaluated in terms of their use as carriers for protein subunit and DNA vaccines. There is an extensive focus on recent literature, the understanding of biological interactions, and relation of this to our present understanding of immunological mechanisms of action. In addition, there is consideration of formulation techniques including emulsification, solvent diffusion, DNA complexation, and entrapment. The diversity of formulation strategies presented is a testament to the exponential growth and interest in the area of vaccine delivery systems. A case study for the application of particulate vaccine carriers is assessed in terms of vaccine development and recent insights into the possible design and application of vaccines against two of the most important pathogens that threaten mankind and for which there is a significant need: Mycobacterium tuberculosis and human immunodeficiency virus. This review addresses the rationale for the use of particulate delivery systems in vaccine design in the context of the diversity of carriers for DNA- and protein-based vaccines and their potential for application in terms of the critical need for effective vaccines. © 2005 by Begell House, Inc.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.

Some aspects of performance at a psychokinetic task

Psychokinetic phenomena are currently anomalous with respect to physics. They are not generally accepted as genuine nor are their possible physical mechanisms understood. It is argued here that a certain class of psychokinetic phenomena, termed "directly detectable" psychokinetic effects, are likely to yield possibly important insights into the physical mechanisms mediating psychokinetic phenomena generally. The current use within parapsychological research of randomly behaving psychokinetic target systems is criticised on several grounds. They are of limited scope for use in delineation of physical mechanisms involved in psychokinesis, and their intrinsic characteristics prevent subjects from utilising their possible capacity to learn to produce larger magnitude effects. It is argued that instrumented directly detectable psychokinetic tasks have characteristics which may allow subjects to treat their psychokinetic ability as akin to a normal skill which can be improved with continued practice, using an experimental paradigm similar to that used in the biofeedback training of physiological functions. The task used in this work was a microscopic form of psychokinetic metal-bending, whereby subjects produce pulse-like electrical outputs in a ceramic piezoelectric element used as psychokinetic target. Subjects were not allowed to touch the target and many effects were obtained under witnessed conditions with subjects situated several metres from it. One pilot and three principal longitudinal training studies were performed with a total of seventeen subjects. Six of the seventeen subjects showed significant improvement in their psychokinetic performance in the training studies, one showed a non-significant increase. The other ten failed to show any convincing signs of psychokinetic output. Three of the successful subjects did not show convincing signs of voluntary control over their effects, three did. Large individual differences were found including different rates of learning and levels of initial and final ability. This research was performed by Julian David Isaacs in preparation for the degree of Doctor of Philosophy and was submitted in 1984.

Melt processable biomaterials for degradable surgical fixation devices

There are currently few biomaterials which combine controlled degradation rates with ease of melt processability. There are however, many applications ranging from surgical fixation devices to drug delivery systems which require such combination properties. The work in this thesis is an attempt to increase the availability of such materials. Polyhydroxybutyrate-polyhydroxyvalerate copolymers are a new class of potentially biodegradable materials, although little quantitative data relating to their in vitro and in vivo degradation behaviour exists. The hydrolytic degradation of these copolymers has been examined in vitro under conditions ranging from `physiological' to extremes of pH and elevated temperature. Progress of the degradation process was monitored by weight loss and water uptake measurement, x-ray diffractometry, optical and electron microscopy, together with changes in molecular weight by gel permeation chromatography. The extent to which the degradation mechanism could be modified by forming blends with polysaccharides and polycaprolactone was also investigated. Influence of the valerate content, molecular weight, crystallinity, together with the physical form of the sample, the pH and the temperature of the aqueous medium on the hydrolytic degradation was investigated. Its progress was characterised by an initial increase in the wet weight, with concurrent decrease in the dry weight as the amorphous regions of the polymer are eroded, thereby producing an increase in matrix porosity. With the polysaccharide blends, this initial rate is dramatically affected, and erosion of the polysaccharide from the matrix markedly increases the internal porosity which leads to the eventual collapse of the matrix, a process which occurs, but less rapidly, in the degradation of the unblended polyhydroxybutyrate-polyhydroxyvalerate copolymers. Surface energy measurement and goniophotometry proved potentially useful in monitoring the early stages of the degradation, where surface rather than bulk processes predominate and are characterised by little weight loss.

Comparative study of dexamethasone and vancomycin release behavior from stimuli-sensitive microgel aqueous dispersions

Stimuli-sensitive microgels of poly(N-isopropylacrylamide-co-acrylic acid) (designated as P(NIPAAm-co-AA)) were prepared through precipitation polymerization. Their capacity to load and release different drugs under different conditions, including physiological, in a controlled manner was analyzed. Two drugs were assayed and compared: dexamethasone and vancomycin. The prepared microgel particles show good thermosensitivity. In addition, the amount of cross-linker used in the preparation of the microgels does not greatly influence the drug-release capability of P(NIPAAm-co-AA)), but the amount of drug used to load the microgels did result in bigger amounts of drug released afterwards. These results imply potential application of prepared stimuli-sensitive microgel dispersions as drug-delivery systems and tissue engineering materials.

G-protein-coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required. © 2014 Cancer Research UK. All rights reserved.

Beyond water homeostasis:diverse functional roles of mammalian aquaporins

Background - Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. Scope of review - AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. Major conclusions - As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. General significance - Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.

Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Liposomal vaccine delivery systems

INTRODUCTION: Liposomes remain at the forefront of drug and vaccine design owing to their well-documented abilities to act as delivery vehicles. Nevertheless, the concept of liposomes as delivery vehicles is not a new one, with most works focusing on their use for the delivery of genes and drugs. However, in the last 10 years a significant amount of research has focused on using liposomes as vaccine adjuvants, not only as an antigen delivery vehicle but also as a tool to increase the immunogenicity of peptide and protein antigens. AREAS COVERED: This paper reviews liposomal adjuvants now in vaccine development, with particular emphasis on their adjuvant mechanism and how specific physicochemical characteristics of liposomes affect the immune response. The inclusion of immunomodulators is also discussed, with prominence given to Toll-like receptor ligands. EXPERT OPINION: The use of liposomes as vaccine delivery systems is evolving rapidly owing to the combined increase in technological advances and understanding of the immune system. Liposomes that contain and deliver immunostimulators and antigens are now being developed to target diseases that require stimulation of both humoral and cell-mediated immune responses. The CAF liposomal system, described in detail in this review, is one liposomal model that shows such flexibility.

Drug delivery strategies for the treatment of Helicobacter pylori infections

Helicobacter pylori is one of the most common pathogenic bacterial infections, colonising an estimated half of all humans. It is associated with the development of serious gastroduodenal disease - including peptic ulcers, gastric lymphoma and acute chronic gastritis. Current recommended regimes are not wholly effective and patient compliance, side-effects and bacterial resistance can be problematic. Drug delivery to the site of residence in the gastric mucosa may improve efficacy of the current and emerging treatments. Gastric retentive delivery systems potentially allow increased penetration of the mucus layer and therefore increased drug concentration at the site of action. Proposed gastric retentive systems for the enhancement of local drug delivery include floating systems, expandable or swellable systems and bioadhesive systems. Generally, problems with these formulations are lack of specificity, limited to mucus turnover or failure to persist in the stomach. Gastric mucoadhesive systems are hailed as a promising technology to address this issue, penetrating the mucus layer and prolonging activity at the mucus-epithelial interface. This review appraises gastroretentive delivery strategies specifically with regard to their application as a delivery system to target Helicobacter. As drug-resistant strains emerge, the development of a vaccine to eradicate and prevent reinfection is an attractive proposition. Proposed prophylactic and therapeutic vaccines have been delivered using a number of mucosal routes using viral and non-viral vectors. The delivery form, inclusion of adjuvants, and delivery regime will influence the immune response generated. © 2005 Bentham Science Publishers Ltd.

Trigger release liposome systems:local and remote controlled delivery?

Target-specific delivery has become an integral area of research in order to increase bioavailability and reduce the toxic effects of drugs. As a drug-delivery option, trigger-release liposomes offer sophisticated targeting and greater control-release capabilities. These are broadly divided into two categories; those that utilise the local environment of the target site where there may be an upregulation in certain enzymes or a change in pH and those liposomes that are triggered by an external physical stimulus such as heat, ultrasound or light. These release mechanisms offer a greater degree of control over when and where the drug is released; furthermore, targeting of diseased tissue is enhanced by incorporation of target-specific components such as antibodies. This review aims to show the development of such trigger release liposome systems and the current research in this field.

Application of genomics, proteomics and metabolomics in drug discovery, development and clinic

Genomics, proteomics and metabolomics are three areas that are routinely applied throughout the drug-development process as well as after a product enters the market. This review discusses all three 'omics, reporting on the key applications, techniques, recent advances and expectations of each. Genomics, mainly through the use of novel and next-generation sequencing techniques, has advanced areas of drug discovery and development through the comparative assessment of normal and diseased-state tissues, transcription and/or expression profiling, side-effect profiling, pharmacogenomics and the identification of biomarkers. Proteomics, through techniques including isotope coded affinity tags, stable isotopic labeling by amino acids in cell culture, isobaric tags for relative and absolute quantification, multidirectional protein identification technology, activity-based probes, protein/peptide arrays, phage displays and two-hybrid systems is utilized in multiple areas through the drug development pipeline including target and lead identification, compound optimization, throughout the clinical trials process and after market analysis. Metabolomics, although the most recent and least developed of the three 'omics considered in this review, provides a significant contribution to drug development through systems biology approaches. Already implemented to some degree in the drug-discovery industry and used in applications spanning target identification through to toxicological analysis, metabolic network understanding is essential in generating future discoveries.