Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding

Banker, Michael J.; Clark, Tracey; Williams, John A.

doi:10.1002/jps.10332

Cited by 282 publications

(203 citation statements)

References 32 publications

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“…Microsomal binding incubations. Microsomal binding in human liver microsomes was performed in triplicate using a 96-well equilibrium dialysis as adapted from a previous described method (Banker et al, 2003). Each dialysis membrane strip was conditioned sequentially for 15 min in deionized water, 30% ethanol, and then 0.1 M potassium phosphate buffer, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

In Silico Modeling of Nonspecific Binding to Human Liver Microsomes

Gao

Yao²,

Mathieu³

et al. 2008

Drug Metab Dispos

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ABSTRACT:Estimation of unbound fraction of substrate in microsomal incubation media is important in accurately predicting hepatic intrinsic clearance and drug-drug interactions. In this study, the unbound fraction of 1223 drug-like molecules in human liver microsomal incubation media has been determined using equilibrium dialysis. These compounds, which include 27 marketed drug molecules, cover a much broader range of physiochemical properties such as hydrophobicity, molecular weight, ionization state, and degree of binding than those examined in previous work. In developing the in silico model, we have used two-dimensional molecular descriptors including cLogP, Kier connectivity, shape, and E-state indices, a subset of MOE descriptors, and a set of absorption, disposition, metabolism, and excretion structural keys used for our in-house absorption, disposition, metabolism, excretion, and toxicity modeling. Hydrophobicity is the most important molecular property contributing to the nonspecific binding of substrate to microsomes. The prediction accuracy of the model is validated using a subset of 100 compounds, and 92% of the variance is accounted for by the model with a root mean square error (RMSE) of 0.10. For the training set of compounds, 99% of variance is accounted for by the model with a RMSE of 0.02. The performance of the developed model has been further tested using the 27 marketed drug molecules with a RMSE of 0.10 between the observed and the predicted unbound fraction values.In drug discovery, the ultimate goal of a medicinal chemist is to rationally design compounds that are safe and efficacious at a marketable dose regimen. Such chemical design efforts frequently rely upon intrinsic clearance (CL int ) and P450 inhibitory potency (IC 50 ) estimates derived from human liver microsomal incubations. With relatively few assumptions, these estimates can be expected to have proportional implications for oral dose requirements and therapeutic windows against pharmacokinetic drug-drug interactions. For example, the classic well stirred model of hepatic clearance can be used to illustrate the directly proportional relationship between CL int and oral dose by eq. 1 (Wagner et al., 1965). Other oral dose determinants include the unbound steady-state average concentration necessary for the desired pharmacologic response (C ss, u ), dose interval (), and fraction of dose absorbed (fa):In addition, by assuming that inhibition of P450 occurs via a competitive mechanism, eq. 2 is a valid and commonly used approach to calculate the therapeutic window against pharmacokinetic drug-drug interactions:Therapeutic window ϭ C ss, u IC 50It has now been well established in the scientific literature that nonspecific binding of compounds into microsomal phospholipids will confound the experimental estimation of CL int and IC 50 by decreasing the fraction of unbound drug (fu mic ) to metabolizing enzymes within the incubation (Kalvass et al., 2001;Margolis and Obach, 2003;Obach, 1997Obach, , 1999Tran et al., 2002). In such ca...

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Section: Methodsmentioning

confidence: 99%

In Silico Modeling of Nonspecific Binding to Human Liver Microsomes

Gao

Yao²,

Mathieu³

et al. 2008

Drug Metab Dispos

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“…The determination of unbound compound was performed in human plasma and in plasma protein buffer solution (45 g/L human serum albumin and 0.7 g/L α 1 acid glycoprotein in phosphate buffer, pH 7.4, respectively) using a 96-well format equilibrium dialysis device with a molecular weight cutoff membrane of 12-14 kDa, as described elsewhere (16,17). The derived samples from each dialysis were then quenched with acetonitrile (ratio, 1:2) containing an internal standard, centrifuged, and quantified by LC-MS/MS.…”

Section: Plasma Protein Binding Determination and Binding To Plasma Pmentioning

confidence: 99%

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

Kratochwil

Meille

Fowler

et al. 2017

AAPS J

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ABSTRACT.Early prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HμREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HμREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.KEY WORDS: in vitro clearance; in vitro liver models; IVIVE; nonlinear mixed-effects modeling.

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“…Older equilibrium dialysis equipment, for example, systems from Dianorm (Munich, Germany), can be laborintensive and time-consuming to use and difficult to automate. Several 96-well format equilibrium dialysis devices have been developed and validated, thus facilitating assay automation (Kariv et al, 2001;Banker et al, 2003;Wong et al, 2009; http://www.HTDialysis.com; http://www.harvardappartus.com). However, these methods still require soaking of membranes and washing and assembly of equipment, thereby increasing the probability of leakage and contamination.…”

Section: Introductionmentioning

confidence: 99%

Control and Measurement of Plasma pH in Equilibrium Dialysis: Influence on Drug Plasma Protein Binding

Curran¹,

Claxton²,

Hutchison³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Past publications have highlighted the influence of postdialysis plasma pH on the measured fraction unbound in plasma (fup). There is disparity in the industry as to which of two main methods is more suitable for controlling postdialysis plasma pH: the use of either a stronger buffer or a CO 2 atmosphere for the incubation. In the current study, it has been found that 10% CO 2 could be too high for the buffering capacities of both 100 mM sodium phosphate (pH 7.40 decreased to pH 6.90 after a 6-h incubation) and plasma (decreased below pH 7.40 after a 6-h incubation). To provide appropriate control over the postdialysis plasma pH, for a range of species, it is proposed that a standard phosphate buffer strength (100 mM) and pH (7.40) in combination with a 5% CO 2 atmosphere be used for equilibrium dialysis. Furthermore, statistically significant differences in fup values obtained with a pH difference of less than 0.32 pH unit have been demonstrated. An acceptance range for postdialysis plasma pH in routine in vitro fup screening assays of pH 7.40 ؎ 0.10 is recommended.

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Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding

Cited by 282 publications

References 32 publications

In Silico Modeling of Nonspecific Binding to Human Liver Microsomes

In Silico Modeling of Nonspecific Binding to Human Liver Microsomes

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

Control and Measurement of Plasma pH in Equilibrium Dialysis: Influence on Drug Plasma Protein Binding

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