Semi-Automated Protein Binding Methodology Using Equilibrium Dialysis and A Novel Mixed-Matrix Cassette Approach

Plise, Emile G.; Tran, Daniel; Salphati, Laurent

doi:10.1002/jps.22188

Cited by 9 publications

(9 citation statements)

References 36 publications

(24 reference statements)

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“…Total matrix and protein free samples were analysed using a matrix matching approach [37] whereby each sample was mixed in a 1:1 ratio with the opposite blank medium (i.e. blank total matrix or blank protein free buffer).…”

Section: Methodsmentioning

confidence: 99%

An in vitro toolbox to accelerate anti-malarial drug discovery and development

Charman

Andreu

Barker

et al. 2020

Malar J

142

183

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BackgroundModelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles.MethodsIonization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC–MS/MS.ResultsBoth solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds.ConclusionsThis is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug–drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

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Section: Methodsmentioning

confidence: 99%

An in vitro toolbox to accelerate anti-malarial drug discovery and development

Charman

Andreu

Barker

et al. 2020

Malar J

142

183

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show abstract

“…A detailed description of the analytical procedure is provide in a recent publication. 9 The comparison of blood-plasma ratio obtained by the described method (which used in vivo blood samples from routine screening studies in rats) with regular in vitro method is provided in Table 1. For in vivo study, the data were obtained using blood samples from three rats, and for in vitro assay the bloodplasma ratio was measured in triplicate by spiking the compounds into blood.…”

Section: Resultsmentioning

confidence: 99%

A Convenient Method to Measure Blood–Plasma Concentration Ratio Using Routine Plasma Collection in In Vivo Pharmacokinetic Studies

Berezhkovskiy¹,

Zhang²,

Cheong³

2011

Journal of Pharmaceutical Sciences

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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%

“…Proposed sample preparation for the equilibrium dialysis (ED)-based brain homogenate method, modified from Plise and coworkers (Plise et al 2010). This method allows eradication of the "matrix" effect often observed in LC-MS/MS when analyzing non-dialyzed vs. dialyzed buffer and homogenate samples, respectively.…”

Section: Fig 101mentioning

confidence: 99%

“…The modifications in the composition of the buffer and brain homogenate could result in a "matrix" effect during subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis (e.g., ion suppression, enhancement of analyte signal) (Van Eeckhaut et al 2009). Mixed-matrix and semi-automated mixed-matrix methods are currently being developed to decrease mass spectrometer run times and reduce the probability of experimental artifacts (Plise et al 2010). For semi-automated mixed-matrix methods with a cassette-based approach, a single matrix is prepared following dialysis by mixing dialyzed plasma and buffer containing different test compounds from the same dialysis plate.…”

Section: Bioanalysismentioning

confidence: 99%

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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-Automated Protein Binding Methodology Using Equilibrium Dialysis and A Novel Mixed-Matrix Cassette Approach

Cited by 9 publications

References 36 publications

An in vitro toolbox to accelerate anti-malarial drug discovery and development

An in vitro toolbox to accelerate anti-malarial drug discovery and development

A Convenient Method to Measure Blood–Plasma Concentration Ratio Using Routine Plasma Collection in In Vivo Pharmacokinetic Studies

Drug Discovery Methods for Studying Brain Drug Delivery and Distribution

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