Drug-protein binding: a critical review of analytical tools

The extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a significant effect on the pharmacokinetics and pharmacodynamics of a drug. It is therefore highly important to estimate drug-binding ability to these macromolecules in the early stages of drug discovery and in clinical practice. Traditionally, equilibrium dialysis is used, and is presented as the reference method, but it suffers from many drawbacks. In an attempt to circumvent these, a vast array of different methods has been developed. This review focuses on the most important approaches used to characterize drug-protein binding. A description of the principle of each method with its inherent strengths and weaknesses is outlined. The binding affinity ranges, information accessibility, material consumption, and throughput are compared for each method. Finally, a discussion is included to help users choose the most suitable approach from among the wealth of methods presented. Figure Range of binding constants (log Ka) assessable by the main separative and non-separative analytical tools used to characterize drug-protein interactions. ED: equilibrium dialysis, UF: ultrafiltration, PAMPA: parallel artificial membrane permeability assay, HPAC/ZE: high-performance affinity chromatography/zonal elution approach, HPAC/FA: high-performance affinity chromatography/frontal analysis approach, ACE: affinity capillary electrophoresis (mobility shift assay), CE/FA: capillary electrophoresis/frontal analysis, Spectro.: spectroscopic assays, ITC: isothermal titration calorimetry, comp.: competition studies, titration: titration studies, DSC: differential scanning calorimetry, SPR: surface plasmon resonance-based assay

La communication de l'enfant polyhandicapé: comment se décline-t-elle dans la prise en charge ?

Ce travail de recherche aborde le thème de la communication chez l’enfant polyhandicapé. Il s’intéresse plus particulièrement aux différentes formes sous lesquelles elle peut se décliner et vise à montrer son incidence sur le bien-être des enfants polyhandicapés. En introduction, vous trouverez les motivations qui m’ont conduite à faire cette recherche. Dans un deuxième temps, vous pourrez apprécier la partie théorique. Les concepts tels que le polyhandicap, les émotions et la communication ont été développés. Cette démarche permet une meilleure compréhension de la problématique étudiée. Pour la recherche, j’ai interviewé trois éducatrices-enseignantes de classes différentes, appartenant à la Fondation Les Perce-Neige. De plus, j’ai effectué des observations dans chacune des trois classes, deux sur le site de La Chaux-de-Fonds et une sur celui de Neuchâtel. Les résultats obtenus lors de l’enquête m’ont permis de tirer certaines conclusions. Elles ont également fait jaillir de nouveaux questionnements. Les éléments suivants ont été mis en avant : • L’enfant polyhandicapé peut communiquer sous différentes formes avec ou sans aide à la communication. • Pour pouvoir s’exprimer, l’enfant a besoin de temps et d’un environnement adéquat. • Pour comprendre l’enfant polyhandicapé, il est nécessaire d’effectuer des observations afin de le connaître. • Entrer en relation avec un enfant polyhandicapé prend du temps. • Pouvoir entrer en relation avec le monde extérieur permet d’atteindre un certain bien-être. Il est évident que les résultats obtenus ne sont pas vérifiables pour tous les enfants polyhandicapés. Tous sont différents et développent des compétences propres à chacun d’eux. C’est aux professionnel·le·s de s’adapter à eux

Analytical strategy to characterize drug-protein interactions: from high throughput to in-depth analysis

La mise sur le marché d'un nouveau médicament est un processus long, complexe et coûteux. Ce n'est pas moins de 15 à 20 ans et un milliard de dollars qui sont nécessaires pour produire un seul médicament. Lors de ce processus, une unique molécule sur 10'000 composés étudiés atteindra ce but. Il a été démontré que l'une des causes majeures du rejet des candidats médicamenteux est un comportement pharmacocinétique inadéquat. Dès lors, la mesure de paramètres physico-chimiques permettant d'estimer, prédire, et comprendre la pharmacocinétique des médicaments est indispensable afin de réduire ce taux de rejet. A ce titre, la liaison aux protéines plasmatiques est un facteur capital à prendre en considération pour comprendre la distribution des médicaments dans l'organisme. Afin de mesurer ce paramètre crucial, une stratégie d'analyse complète permettant d'évaluer la liaison aux protéines plasmatiques à différents stades du processus de développement du médicament a été mise en place. Dans un premier temps, lorsque le nombre de composés à analyser est important, une méthode rapide et générique en chromatographie liquide a été mise au point. Plus tard dans le développement du médicament lorsque le nombre de composés diminue inversement au besoin en informations détaillées, une méthode d'électrophorèse capillaire ainsi qu'une méthode basée sur le phénomène de résonance des plasmon de surface ont été développées

Global analytical strategy to measure drug–plasma protein interactions: from high-throughput to in-depth analysis

The selection of drug candidates with improved pharmacokinetics is essential to reduce the attrition rates during drug development and represents one of the big challenges faced by the pharmaceutical industry. Plasma protein binding (PPB) is an important parameter with significant implications for in vivo drug performance. Today, the most widely used techniques for PPB measurement in the pharmaceutical community are equilibrium dialysis (ED) and ultrafiltration (UF). However, these techniques have some limitations. Thus, we emphasize an alternative strategy, based on a global, new and easy-to-follow methodology, to screen and perform determination of PPB, using orthogonal techniques (i.e. liquid chromatography (LC), capillary electrophoresis (CE), surface plasmon resonance (SPR) based biosensor). We anticipate that the increased knowledge gained through this strategy will lead to improved drug candidates

Characterization of drug-protein interactions by capillary electrophoresis hyphenated to mass spectrometry

The demand for analytical techniques to evaluate and measure drug–plasma protein interactions continues to increase. The binding of drugs to plasma proteins is an important parameter to determine during the drug development process because it impacts both pharmacokinetics and pharmacodynamics. Among the numerous methods that have been proposed to perform such studies, CE in frontal analysis mode (CE/FA) is attractive because it consumes a relatively low amount of samples, is fast, and enables analyses under near-physiological conditions. Most CE/FA applications have been performed with UV detection and often lack sensitivity. In this study, CE was hyphenated to MS to enhance the sensitivity of the method and to evaluate strong drug–plasma protein interactions. To adapt the previously developed CE/FA-UV method to CE/FA-MS, different parameters were considered, such as the buffer composition, the rinsing step, and the ESI and MS parameters. The most critical aspect involved obtaining stable MS signals. Good results were achieved due to careful optimization of the ESI and MS parameters, among which the sheath liquid composition appeared to be the most significant. Interactions between six drugs and 1-acid glycoprotein and three drugs and BSA, including basic, neutral, and acidic drugs, were measured with the optimized CE/FA-MS method. The obtained affinity constants ranged from 1·10−4 M−1 to 2·10−5 M−1 and were in good agreement with the results that were obtained by CE/FA-UV and equilibrium dialysis

Drug-protein binding: a critical review of analytical tools

The extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a significant effect on the pharmacokinetics and pharmacodynamics of a drug. It is therefore highly important to estimate drug-binding ability to these macromolecules in the early stages of drug discovery and in clinical practice. Traditionally, equilibrium dialysis is used, and is presented as the reference method, but it suffers from many drawbacks. In an attempt to circumvent these, a vast array of different methods has been developed. This review focuses on the most important approaches used to characterize drug-protein binding. A description of the principle of each method with its inherent strengths and weaknesses is outlined. The binding affinity ranges, information accessibility, material consumption, and throughput are compared for each method. Finally, a discussion is included to help users choose the most suitable approach from among the wealth of methods presented

High performance affinity chromatography (HPAC) as a high-throughput screening tool in drug discovery to study drug–plasma protein interactions

Drug–plasma protein binding is an important parameter that, together with other physicochemical properties such as lipophilicity and pKa, 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 α1-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

Comparison of various silica-based monoliths for the analysis of large biomolecules

In the present study, three types of silica-based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide-pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first-generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa)but very close with smaller ones (1.3–5.8 kDa). In contrast, the wide-pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide-pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica-based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa)

Improvement of a capillary electrophoresis/frontal analysis (CE/FA) method for determining binding constants: Discussion on relevant parameters

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