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Bicalutamide (Casodex®) is a non-steroidal anti-androgen drug used in the treatment of prostate cancer, which represents the second most common malignancy diagnosed in men worldwide. In this work, we analyze the ability of some novel bicalutamide analogs to bind the androgen receptor, by using an optical biosensor. Androgen receptor was covalently immobilized on a carboxy methyl dextran matrix. The immobilized receptor chip was then used for the binding experiments of the bicalutamide analogs. The (R)-bicalutamide dissociation constant was in good agreement to the value reported in literature obtained by using radiolabeled targets. Most of the new synthesized compounds showed higher androgen receptor binding level, when compared to the reference. Our results clearly indicate that the surface plasmon resonance (SPR) technique offers many advantages with respect to other available technologies in terms of studying biomolecular interactions. Moreover, this study provides an effective methodology for determining the binding affinity of novel chemical entities for the isolated androgen receptor, thus excluding possible off-target interactions occurring in conventional cell-based techniques., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Drug-plasma protein binding is an important parameter that, together with other physicochemical properties such as lipophilicity and pK(a), greatly influences drug absorption, distribution, metabolism, and excretion (ADME). Therefore, it is important for pharmaceutical companies to develop a rapid screening assay to examine plasma protein binding during the early stages of the drug discovery process. Human serum albumin (HSA) and α(1)-acid glycoprotein (AGP) are the most important plasma proteins that are capable of binding drugs. In this work, an automated and high-throughput (<3 min/compound) strategy was developed using high performance affinity chromatography (HPAC) with commercial HSA and AGP columns to evaluate drug-plasma protein interactions for drug screening. A generic gradient was used throughout the study to separate drugs that were weakly and tightly bound to HSA and AGP. To accelerate the analysis time, the system was calibrated in a single run by pooling reference compounds without overloading the column. For both HSA and AGP studies, the developed methods were successfully transferred from HPAC-UV to HPAC-MS with single quadrupole MS detection and ammonium acetate, pH 7.0 as a volatile mobile phase. The MS detection enhanced the sensitivity, selectivity, and throughput of the method by pooling unknown compounds. For HSA analyses, the binding percentages obtained using HPAC were well correlated with the binding percentages from the literature. This method was also able to rank compounds based on their affinity for HSA. Concerning the AGP analyses, the quality of the correlation between the binding percentages obtained in HPAC and those from the literature was weaker. However, the method was able to classify compounds into weak, medium, and strong binders and rank compounds based on their affinity for AGP., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Drug-plasma protein interactions have a significant impact on both pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (pharmacological effects). Therefore, it is of high interest to evaluate this binding during the drug development process. Capillary electrophoresis (CE) is an interesting analytical tool for drug-protein binding characterization because it consumes a relatively low amount of reagents and enables assays that can be carried out under near-physiological conditions. The most interesting mode of CE for the study of biomolecular interactions is CE/frontal analysis (CE/FA). However, some confusion in how to conduct CE/FA experiments has emerged in the literature. The present study examines, using research into drug-albumin interactions as an example, the most important steps to take into consideration when building up new CE/FA binding assays. These include the following: choosing the buffer and applied voltage; evaluating protein adsorption onto the capillary wall; choosing the injection volume; choosing the drug and protein concentrations; and, finally, verifying the co-migration of the protein and drug-protein complex. The experimental part of the present report can serve as a checklist for developing the key parameters that need to be addressed for successful and reliable interaction studies. In a second time, short-end injection was used to enhance throughput. The strengths of the binding constants (K(a)) for nine selected drugs (basic, neutral, and acidic substances) to albumin, which is the most important plasma protein, were from logK(a) 2.9 to 5.4. These values were compared to those obtained with validated methods and good agreement was achieved., (Copyright 2010 Elsevier B.V. All rights reserved.)

A liquid chromatography method for the characterization of base deactivated columns was investigated in a collaborative study involving six laboratories. This work was carried out on two chromatographic supports (Xterra RP 18 and Symmetry Shield). Different cooling systems, namely water bath and air oven, were tested and it was shown that column thermoregulation did not significantly influence chromatographic data. In order to control the mobile phase composition, the latter was prepared by weight rather than volume. Thanks to the injection of a set of selected neutral compounds, extra-column effects were evaluated in each of the participating laboratories. The results showed that chromatographic supports tested in different laboratories and following the same test protocol could be effectively compared.

This paper describes a new spectrofluorimetric assay for continuously monitoring the enzymatic hydrolysis of medicinal esters. The procedure is based on the stoichiometric quantity of protons generated by the hydrolysis of the substrate, which produces changes in the fluorescence of a pH-sensitive dye. The pH indicator, 2', 7'-bis(car-boxyethyl)-5(6)-carboxyfluorescein, was selected due to its favourable pKa for studies under physiological conditions. Moreover, the presence of a domain in the spectra (< 442 nm) where fluorescence intensities are independent of pH allows measurements of wavelength ratios that cancel artifacts and lower sample-to-sample variability. The indicator did not affect the catalytic activity of purified hog liver carboxylesterase or human serum albumin. This assay is easy to perform and appears to be especially useful for studying enzymatic reactions with half-lives of the order of minutes or hours.