Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography–tandem mass spectrometry

Fung, Eliza N.; Chen, Yung‐Hsiang; Lau, Yau Yi

doi:10.1016/s1570-0232(03)00564-6

Cited by 75 publications

(40 citation statements)

References 23 publications

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“…At present, the dialysis-based assay is considered to be an accurate method for the rapid estimation of the free fraction of drugs in brain (f u,brain ). However, the equilibrium dialysis method is still a time-consuming assay with limited sample throughput capacity, and although improvements have been achieved by sample pooling approaches (Fung et al, 2003;Wan and Rehngren, 2006;Wan et al, 2007), the throughputs of dialysis-based methods are still limited by long equilibration times (4 -6 h) and the readily available supply of brain homogenate. As a result, the development of a matrix-free, high-throughput method for assessing f u,brain would be highly desirable.…”

Section: Introductionmentioning

confidence: 99%

Brain Tissue Binding of Drugs: Evaluation and Validation of Solid Supported Porcine Brain Membrane Vesicles (TRANSIL) as a Novel High-Throughput Method

Longhi

Corbioli²,

Fontana³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Estimating the unbound fraction of drugs in brain has become essential for the evaluation and interpretation of the pharmacokinetics and pharmacodynamics of new central nervous system drug candidates. Dialysis-based methods are considered to be accurate for estimating the fraction unbound in brain; however, these techniques are hampered by a low throughput. In this study, we present a novel, matrix-free, high-throughput method for estimating the unbound fraction, based on a sample pooling approach combining the TRANSIL brain absorption assay with liquid chromatography-mass spectrometry. The base measurement of the TRANSIL approach is the affinity to brain membranes, and this method is used directly to predict the free fraction in brain.

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Section: Introductionmentioning

confidence: 99%

Brain Tissue Binding of Drugs: Evaluation and Validation of Solid Supported Porcine Brain Membrane Vesicles (TRANSIL) as a Novel High-Throughput Method

Longhi

Corbioli²,

Fontana³

et al. 2010

Drug Metab Dispos

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“…Rat plasma protein binding was determined by equilibrium dialysis. Dialysis membranes (molecular weight cutoff of 5000; NBS Biologicals Ltd., Huntingdon, Cambridgeshire, UK) were prepared by soaking in the dialysis buffer (0.1 M phosphate buffer) for a minimum of 1 h. Stock solutions of compounds in dimethyl sulfoxide were prepared and added to the plasma to give a final concentration of 5 M. Mixtures of up to 10 compounds per dialysis cell were dialyzed for 18 h at 37°C, in accordance with established literature methods (Fung et al, 2003;Testa et al, 2006). The plasma and buffer were analyzed by high-performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS) on a Waters Premier XE triple quadrupole mass spectrometer.…”

Section: Methodsmentioning

confidence: 99%

Prediction of Efficacious Inhalation Lung Doses via the Use of In Silico Lung Retention Quantitative Structure-Activity Relationship Models and In Vitro Potency Screens

Cooper

Potter²,

Luker³

2010

Drug Metab Dispos

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ABSTRACT:Lung concentrations of a drug are expected to drive the pharmacodynamic response to local inflammation after inhalation delivery, and the only way of determining the efficacious dose has been to measure it directly in animal models. In this study, we present a method to predict efficacious lung doses after inhalation in a rat lipopolysaccharide challenge model from in silico predictions of lung concentration and in vitro measurements only. A quantitative structure-activity relationship (QSAR) model, based on calculated physical properties predicted the partitioning of 34 compounds between lung and plasma. Because it was observed that lung/ plasma partitioning correlated with lung concentration, it was possible to use this relationship to predict lung concentration at a given dose and time point. Based on the pharmacokinetic-pharmacodynamic (PKPD) relationship observed, a minimal free lung concentration relative to potency to drive significant inhibition of neutrophilia was established. By using predicted lung concentrations, measured fraction unbound in plasma, and cellular potency, it was possible to estimate an inhaled lung dose that would be expected to achieve this target exposure. These predictions were made for 23 compounds, which were not part of the original QSAR training set, and all except one were predicted to within 3-fold of their measured values. This novel approach shows that by understanding PKPD relationships and drivers for lung affinity after inhalation dosing, it is possible to estimate in vivo lung doses required for efficacy. This methodology provides a useful screening tool to rank candidate compounds and minimizes the use of extensive animal testing.

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“…The method rapidly became standard and it is currently widely used for estimation of f u,brain for a large number of chemically diverse compounds (Summerfield et al 2006;Wan et al 2007;Di et al 2011;Friden et al 2011;Longhi et al 2011). The need for protein binding data in combination with the large number of compounds created from combinatorial chemistry has stimulated the development of a novel cassette-based pooling approach which allows simultaneous assessment of f u,brain or f u,plasma for more than five compounds per sample (Fung et al 2003;Wan et al 2007;Plise et al 2010;Longhi et al 2011).…”

Section: Equilibrium Dialysismentioning

confidence: 99%

Drug Discovery Methods for Studying Brain Drug Delivery and Distribution

Loryan

Hammarlund‐Udenaes

2013

Drug Delivery to the Brain

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Methods used in drug discovery laboratories for assessing the delivery of small molecules to the brain have changed significantly in recent years. There is now more focus on measuring or estimating target unbound drug concentrations in the brain and evaluating the quantitative aspects of drug transport across the bloodbrain barrier (BBB). The techniques for investigation of the rate and extent of BBB transport of new chemical entities (NCEs) are discussed in this chapter. Combinatory methodology for rapid mapping of the extent of brain drug delivery via assessment of the unbound drug brain partitioning coefficient is presented. The chapter also explains the procedures for approximation of subcellular distribution of NCEs, particularly into the lysosomes. The principles, technical issues, advantages, and potential applications of techniques for evaluation of intra-brain distribution, i.e., equilibrium dialysis-based brain homogenate and brain slice methods, are described. The assessment of extent of BBB transport and intracellular distribution of NCEs, the identification of intra-brain distribution patterns, and their integration with pharmacodynamic measurements are valuable implements for candidate evaluation and selection in drug discovery and development. Abbreviations A brainAmount of drug in brain tissue AUC tot,brain Area under the total brain concentration-time curve AUC tot,plasma Area under the total plasma concentration-time curve BBB Blood-brain barrier

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Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography–tandem mass spectrometry

Cited by 75 publications

References 23 publications

Brain Tissue Binding of Drugs: Evaluation and Validation of Solid Supported Porcine Brain Membrane Vesicles (TRANSIL) as a Novel High-Throughput Method

Brain Tissue Binding of Drugs: Evaluation and Validation of Solid Supported Porcine Brain Membrane Vesicles (TRANSIL) as a Novel High-Throughput Method

Prediction of Efficacious Inhalation Lung Doses via the Use of In Silico Lung Retention Quantitative Structure-Activity Relationship Models and In Vitro Potency Screens

Drug Discovery Methods for Studying Brain Drug Delivery and Distribution

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