Pharmacokinetics, Pharmacodynamics and Drug Metabolism
On the Accuracy of Determination of Unbound Drug Fraction in Tissue using Diluted Tissue Homogenate

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ABSTRACT

The unbound drug fraction in tissue, fut, is commonly measured in vitro using the diluted tissue homogenate. An appraisal of the calculation procedure that is routinely applied to obtain fut is presented. An accurate detailed calculation that takes into account the drug protein binding in tissue extracellular water and the pH difference between extra- and intracellular water is considered. It turns out that for neutral compounds, the routine calculation provides fut quite accurately. Though for acidic compounds, the routine calculation can considerably underestimate fut up to 1.8-fold for monoprotic and up to 2.4-fold for diprotic ones, whereas for basic compounds, fut can be substantially overestimated up to 1.9-fold for monoprotic and up to 4.3-fold for diprotic ones. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:1909–1916, 2012

Section snippets

INTRODUCTION

Distribution of drug in tissues is one of the features that determine its concentration profile and pharmacological activity. At steady state, it can be characterized by the value of unbound drug fraction in each tissue, fut, which is defined asfut=CutCt

where Cut is the concentration of unbound drug in tissue extracellular water and Ct is the total tissue concentration. At equilibrium, the unbound drug concentration in plasma, Cup, is equal to Cut because the pH of plasma and tissue

RESULTS

To derive the equation for fut, we need to consider the equilibrium drug distribution between tissue compartments, which are the extra- and intracellular water, and membrane and connective tissues (considered as a single compartment).1,2 Tissue extracellular water has pH 7.4, while intracellular water has pH 7.0. At equilibrium, the concentrations of unbound neutral drug in extracellular water, Cnu,EW, is the same as unbound neutral drug concentration in intracellular water, Cnu,IW, so thatCu,EW

DISCUSSION

A possible underestimation of fut for acidic compounds and overestimation for basic ones occurs because of pH difference between extra- and intracellular water. Acidic compounds tend to stay in the extracellular water, whereas the basic compounds distribute more into intracellular water (due to its lower pH). This selective distribution of compounds does not exist in tissue homogenate, so that the measured fut,h may not adequately provide the value of fut when a simplified calculation (Eq. 4)

APPENDIX 1

Tissue homogenate can be considered as comprised of water at pH 7.4 and membrane–connective tissue constituent. The water of homogenate includes both the extra- and intracellular water from tissue and the added water for dilution. Thus, its volume, VW*, is equal toVW*=VEW+VIW+Vo+Vadd

It is assumed here that the tissue was perfused with water to remove blood and consequently to eliminate a possible error in measurement of fut,h because of the distribution of compound into residual blood. Protein

List of Notations

  • AUCp, AUCt—the areas under the drug concentration-time curves for plasma and tissue, respectively;

  • Cp—total drug plasma concentration;

  • Ct—total tissue concentration;

  • Ct,h—total drug concentration in tissue homogenate;

  • Cut,h—unbound drug concentration in tissue homogenate;

  • Cut—concentration of unbound drug in tissue extracellular water;

  • CEW, CIW—the total drug concentrations in extra- and intracellular water, respectively;

  • Cnu,EW,Cnu,IW—the concentrations of unbound neutral drug in tissue extra- and

REFERENCES (17)

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