Application of the Fluorescent Probe 1-Anilinonaphthalene-8-Sulfonate to the Measurement of the Nonspecific Binding of Drugs to Human Liver Microsomes

J Mclure, Donald Birkett, David Elliot, Andrew Williams, Andrew Rowland, John Miners

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    Abstract

    The fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) in the presence of human liver microsomes (HLMs) is altered by drugs that bind nonspecifically to the lipid bilayer. The present study characterized the relationship between the nonspecific binding (NSB) of drugs to HLMs as measured by equilibrium dialysis and the magnitude of the change in baseline ANS fluorescence. Fraction unbound in incubations of HLMs (f u(mic)) was determined for 16 drugs (12 bases, 3 acids, and 1 neutral) with log P values in the range 0.1 to 6.7 at three concentrations (100, 200, and 500 μM). Changes in ANS fluorescence induced by each of the drugs in the presence of HLMs were measured by spectrofluorometry. Values of f u(mic) determined by equilibrium dialysis ranged from 0.08 to 1.0. Although NSB of the basic drugs tended to increase with increasing log P, exceptions occurred. Basic drugs generally caused an increase in ANS fluorescence, whereas the acidic and neutral drugs resulted in a decrease in ANS fluorescence. There were highly significant (p < 0.001) linear relationships between the modulus (absolute value) of the increment/decrement in ANS fluorescence and both f u(mic) (r = 0.90 to 0.96) and log(1 - f u(mic)/f u(mic)) (r = 0.85 to 0.92) at the three drug concentrations. Agreement between measured f u(mic) and that predicted by ANS fluorescence was very good (<10% variance) for a validation set of six compounds. The ANS fluorescence method provides an accurate measure of the NSB of drugs to HLMs. Physicochemical determinants other than log P and charge type influence the NSB of drugs to HLMs.

    Original languageEnglish
    Pages (from-to)1711-1717
    Number of pages7
    JournalDrug Metabolism and Disposition
    Volume39
    Issue number9
    DOIs
    Publication statusPublished - Sep 2011

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