Combinatorial Chemistry & High Throughput Screening - Volume 13, Issue 6, 2010

Analytical methods widely utilize the knowledge of all the other disciplines such as biology, pharmaceutical chemistry, pharmaceutical technology, biotechnology and many other fields. Thereby, they provide all these with data on the character and amount of chemical species contained in natural and man-made objects and on the distribution of these species in space and time. Work in Analytical Chemistry is divided into two interrelated parts: The methodological part includes the study of physico-chemical processes from the analytical point of view, construction of the related instrumentation and the development and optimization of analytical methods. The practical part adjusts these analytical methods to the necessities of the users. The various analytical methods that permit the determination of drugs in their dosage forms, raw material, biological media etc, and also their metabolites to be separated, identified and quantitatively assayed are briefly reviewed. The current significance of separation, electrochemical, spectrophotometric and other methods is highlighted, as well as the limits of trace assays. The sensitivity of the analytical assay has a direct impact on the validity of the pharmacokinetic model which is built up from plasma concentration data. The precision and accuracy of the assay is also important, and it is not always straightforwardly estimated. A new significant parameter is the speed of analysis, and the resulting massive production of analytical data. New drugs coming from biotechnology, and their dosage forms, like targeted drugs, may produce new analytical problems in the future. In addition to other analytical methods, the use of electrochemical methods to gain key information about drug molecules and their mechanism of action is getting one of the important ways in drug discovery. Although electrochemical data do not give absolute relationship with biological activity, due to in vitro conditions it is still possible to consider that the oxidation mechanisms taking place at the electrode and in the body share related principles as a result of the existing similarities between electrochemical and biological reactions. Applications of electrochemical techniques to redox-active drug development and mechanistic studies are one of the recent interests in drug discovery. It should be noticed that many vital physiological processes are depending on oxido-reduction reactions. So, it is easy to find connections between electrochemical and biological reactions regarding electron transfer pathways. Pharmaceutical Sciences have contributed to drug development, synthesis, formulations and analysis through extensive studies in drug assays. Analytical and pharmaceutical chemists and pharmaceutical scientists play important roles in monitoring the drugs in their dosage forms and biological samples. From the viewpoints mentioned above, the title of this special issue, “Present Applications of Analytical Methods: Prospects for High Throughput Screening of Pharmaceutically Active Compounds”, was chosen so as to ask analytical chemists and pharmaceutical scientists to appreciate their great roles in pharmaceutical science. Three issues feature 12 Reviews and 20 Original Papers. In the first issue: resent developments of separation techniques such as capillary electrophoresis, liquid chromatography, high throughput screening methodologies, chiral analysis and spectrophotometric methods and their biological and pharmaceutical applications are presented. Second issue mainly contains current progresses in DNA biosensors, sensors, electroanalytical methods and their applications to the pharmaceuticals and biological samples, new electrode materials such as carbon nanotubes, boron doped diamond, etc. Finally, the third issue represents all important sensors, analytical and electroanalytical methods. In the last issue chiral, chromatographic, electroanalytical, sensor, atomic spectroscopic and colorimetric techniques and their applications were reported. The purpose of this special issue will be to serve as a guide to what analytical methods bring to analytical and medicinal chemistry and other pharmaceutical sciences as well as briefly review their role in drugs and the new developments and validation of assay methods of pharmaceutically active compounds. This special issue is multidisciplinary. Also recent developments of application, evaluation and validation of analytical methods are focused by key topics in drug developments and analysis by assessment of the distinguished authors of this special issue. Thus, I expect this special issue will assist readers to find out new information and to encourage them to contribute more to recent development on drug development and its analysis using different methods.I hope that the reader will find a number of topics of interest, and that additional new ideas will emerge from this special issue. I would like to thank to all of the authors for their excellent contributions, and the Editor-in-Chief of Combinatorial Chemistry & High Throughput Screening for his kind invitation to act as guest editor for this special issue.

Enzyme Immobilized Magnetic Nanoparticles (EMNPs) were injected and magnetically retained, as a microreactor, in the capillary of a capillary electrophoresis (CE) setup with UV detection. The enzyme horseradish peroxidase (HRP) was chemically immobilized onto commercially available magnetic 300 nm diameter nanoparticles. Paracetamol (acetaminophen: N-acetyl-p-aminophenol), a common analgesic drug, was used as model drug compound. The enzymatic reaction was studied in-line by CE in 12.5 mM phosphate buffer pH 7.4 containing 20 mg/ml sulfated-β- cyclodextrin and 0.1 mM hydrogen peroxide. By means of the developed setup, the apparent Michaelis Menten constant between HRP and acetaminophen (APAP) was determined as Km app = 53±5 μM. This approach was found to be of interest for enzyme kinetics studies with short time resolution condition. Based on our results and from the literature data, it was possible to infer that the in-line generated product was an APAP dimer. Higher enzyme immobilized beads loading in the CE setup generated the APAP dimer with two additional minor peaks likely attributing to APAP trimer and tetramer. N-acetyl- p-benzoquinone imine (NAPQI) was not generated during APAP short time migration through the in-line microreactor.

Sequential injection analysis (SIA) has been employed as the platform for the development of a high throughput automated assay for glutathione (GSH) in nutrition supplements. The proposed assay is based on the on-line formation of a UV-absorbing thioacrylate derivative (δmax = 285 nm) upon reaction of thiol with the new reagent ethylpropiolate (EP). This is the very first application of this reagent for the determination of GSH either in batch or continuous flow modes and the first SI method for the determination of GSH in pharmaceutical supplements. The various variables that typically affect such a protocol were studied, while thorough validation experiments according to established standards proved the reliability of the assay for the intended applications. As many as 100 sample injections were enabled per hour.

In a variety of countries, juvenile idiopathic arthritis (JIA) is the most common cause of chronic arthritis in childhood, yet its etiology is still unknown. In recent years, etanercept, an effective inhibitor of tumor necrosis factor alpha (TNF-α), was used as an alternative in certain oligoarticular JIA patients resistant to conventional nonsteroidal antiinflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and corticosteroid therapies, and it resulted in sustained improvement in JIA symptoms. This pilot study explores the alterations of specific panels of cytokines and protein profiles in plasma for two Taiwanese pediatric cases with diagnosed enthesitis-related arthritis (ERA), a type of JIA. The patients were studied before and after taking etanercept alone, using a high-content screening approach employing membrane-based human cytokine antibody microarray and the conventional two-dimensional gel electrophoresis (2-DE) proteomic technique. Specifically, 2-DE in combination with mass spectrometry (MALDI-MS) revealed the functional roles of plasma proteins associated with the regulation of immune responses during short-term etanercept treatment of children with ERA. Our study shows that this biotherapy improved clinical ERA manifestations through the regulation of inflammatory mediators, including several cytotoxic cellular cytokines (IL-2/IFN-γ), chemokines (MCP-1), and growth factors (GRO) that affect the expression of specific acute phase proteins such as haptoglobins, immunoglobulin A, and fibrinogen-γ chain. Meanwhile, an up-regulation of antithrombin chain I, vitamin-D binding protein (VDBP), and the various apolipoproteins was also observed after the administration of etanercept in both studied children. These results may be interpreted as the relevant predictive biomarkers of therapeutic responses to etanercept. They suggest that etanercept, which is still rarely used in Taiwan, is a viable treatment for JIA patients, without adverse health effects and increased risk of secondary infections.

Advances in the design of automated compound storage systems have made it possible to store large collections of research compounds in individual single-use aliquots dissolved in dimethyl sulfoxide and rapidly retrieve a specific group off them. This 'cherry-picking' approach offers researchers the opportunity to request large numbers of compounds desired for testing without having to also retrieve all the other compounds stored on the same rack or plate. This makes it possible to meet the increasing demand for samples from High Throughput Screening and Therapeutic Area teams without adding staff to dispense from powder each time, without the constraints imposed by storing in solvated compounds in fixed-well 96- or 384-way plates, and without sacrificing sample quality or shelf life by storing at room temperature. We describe how this approach has been implemented at Abbott Laboratories' central compound repository to provide smaller amounts of more compounds faster and with high quality. In doing so, we have been able to better support the innovation of our Drug Discovery colleagues.

A gastro-resistant (enteric coated) etodolac tablet dosage form, was evaluated by using in vitro and in vivo methods. In vitro drug release studies have shown that enteric coated tablet dosage form protects the drug from being released under conditions mimicking stomach to small intestine transit. The gastro-intestinal transit of radiolabeled 300 mg enteric coated etodolac tablets in six healthy, fed and fasted state voluntrees was monitored using external gamma camera. Dosage form position was reported at several time intervals. Etodolac serum concentrations were determined from serum samples drawn over 420 min following dosing. Differences in gastro-intestinal transit between fed and fasted volunteers had little effect on etodolac bioavailability. AUC, Tmax and C max values were calculated for both types of studies.

A sequential injection analysis system was selected for the simultaneous determination of L- and D-T4 in raw material and pharmaceutical products, because of the highest precision and accuracy, the lower consumption of sample and buffer, and the flexibility of selecting different buffers accordingly with the type of sensors utilized as detectors. An amperometric biosensor based on D-amino acid oxidase was used as detector for D-T4 and an immunosensor based on anti-L-T4 was used as detector for L-T4. The SIA/biosensors system can be used reliably on-line in synthesis process control, for the simultaneous assay of L- and D- T4 with a frequency of more than 30 samples per hour.

In this study, a simple, rapid, cost-effective, and sensitive reversed-phase high-performance liquid chromatographic method has been developed and validated for the analysis of moxifloxacin in plasma. The chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.) connected to a Phenomenex C18 column (4 mm x 3.0 mm i.d.) using a mixture of acetonitrile: 15 mM citrate buffer (pH 3) (23:77, v/v) as the mobile phase with isocratic system at a flow rate of 1 mL/min. Fluorescence detection was employed with excitation at 290 nm and emission at 500 nm. Lomefloxacin was used as internal standard. Plasma samples were prepared with addition of acetonitrile only. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The results of the validation parameters were: linearity range, 3-6000 ng/mL (R2 = 0.9994); mean recovery, 100.48 %; limit of quantification, 5 ng/mL; limit of detection, 1 ng/mL; and intra- and inter-day precision less than 3.2% and 5.1%, respectively. The robustness of the method was evaluated and confirmed with fractional factorial design. After validation studies, the method was applied in order to conclude the effects of pregnancy on postoperative pharmacokinetic profiles of moxifloxacin. For this aim, moxifloxacin was given to non-pregnant women (n=9) and caesarean-sectioned women (n=6) as a single intravenous dose (400 mg Avelox® infusion). Plasma samples were analyzed in order to compare pharmacokinetic profiles of pregnants and non-pregnants. Peak serum concentrations of non-pregnant and caesarean-sectioned women at the arterial port after the infusion were 4.95 ± 1.50 and 1.56 ± 0.16 μg/mL, respectively. The mean elimination half-life, volume of distribution and calculated area under the concentrationtime curve (AUC)0-∞ were 5.54 ± 0.73 h, 65.58 ± 6.30 L and 49.95 ± 6.30 μg h/mL for non-pregnant women and 3.50 ± 0.37 h, 215.85 ± 24.87 L and 10.53 ± 0.66 μg h/mL for caesarean-sectioned women, respectively. These results indicated that pregnancy has a significant effect on the pharmacokinetic profiles of moxifloxacin.

In recent years, a trend of change has been observed within pharmaceutical industry. As modern drug discovery has reached a remarkable level of complexity and drugs need to be discovered, developed and produced against strict timelines and within cost- and regulatory constraints, industry seeks “lean” solutions to increase productivity. Among them, increasing the sample throughput of the ever-growing number of necessary (routine) analyses has become a popular target to cut precious time. For the last thirty years, High-Performance Liquid Chromatography (HPLC) has been the leading technology when it comes to various analyses in pharmaceutical industry; however, its necessity of serial analyses taking typically 10-45 min has been a sample throughput-limiting barrier. Lately, the fundamentals of HPLC have been exploited to raise new technologies that can speed up analyses to ground breaking limits, without compromising separation efficiency. This paper reviews some promising technologies, i.e. totally porous sub-2μm particles accompanied by pressures up to 1000 bar (Ultra-Performance Liquid Chromatography or UPLC), fused-core particle technology, monolithic supports and High Temperature Liquid Chromatography (HTLC), having the potential to take LC to the next level in pharmaceutical industry. As each analytical method has its own demands, the advances of the above technologies are discussed for different applications in pharmaceutical analysis where high throughput analysis can be meaningful, i.e. in a drug discovery and development setting, and in quality operations. Both chemical and biological pharmaceuticals are considered. We discuss the perspectives of these technologies and their realizations up to now in high throughput pharmaceutical analysis.

In this review paper, the high throughput potential of some novel chromatographic modes is surveyed. The modes are Hydrophilic Interaction Liquid Chromatography (HILIC), Supercritical Fluid Chromatography (SFC), and Polar Organic Solvent Chromatography (POSC). Their high throughput potential will be discussed in three domains, i.e. drug discovery, bio-analysis in clinical drug development, and quality control (QC) testing, and is illustrated with some examples.

Over the years, many different high throughput screening technologies and subsequently follow-up methodologies have been developed. All of these can be categorized, for example according to measurement of analyte classes, assay mechanisms, readout principles, or screening of drug target classes. When categorized according to drug target class, assay formats can be subdivided into early hit stage assays (usually ligand-binding based) that are often straightforward and robust up to analysis of final cellular effects exerted by ligands. The latter do not only provide higher content data but also represent anticipated effects in the body more closely. These assay formats, however, are often also elaborate, non-robust, and very time consuming to conduct but become pivotal when going from the hit-to-lead discovery stage. Looking at the hit-to-lead process, new assay possibilities in terms of measured effects and readout principles are continuously reaching the screening arena. Furthermore, at the (early-) lead discovery stage other targets also have to be evaluated to study e.g. target selectivity and ADME(T). This review discusses many of the different assay formats used in the hit-to-lead discovery phase sorted by their use for screening major drug target classes for small molecule drug discovery. The receptor drug targets in this review are subdivided into GPCRs, nuclear receptors and ion channels, while for the enzyme drug targets the important protein kinases, proteases and the drug metabolism enzymes (CYPs; mainly important for drug metabolism and drug-drug interactions) are discussed in more detail. For every drug target, different assay formats used to analyze ligand mediated effects at specific points in the drug target's respective signal transduction cascade(s) are looked at. More specifically, assay methodologies for ligand binding and second messenger formation up to signal transduction cascades and analysis of eventual (cell based) effects are described. Furthermore, special attention is paid to less traditional (non-platereader based) state-of-the-art screening approaches such as LC-MS, microarrays, microfluidics and sensor based technologies.

Nano scale chiral separation is essential and has become the demand of current century in drugs development, proteomic, genomic and environmental sciences. Some drugs remain in our body tissues for several weeks at nano scale, which are out of the domain of detection by conventional analytical methods. Similarly, the analyses and detection of xenobiotics; present at nano or low levels; are essential for our health. The present review describes the state-of-art of nano scale chiral separations by nano liquid chromatography (NLC) and nano capillary electrophoresis (NCE). The optimization strategies of chiral separations have been also discussed. Furthermore, efforts were made to discuss the mechanisms of chiral separations in NLC and NCE.