DETERMINATION OF PROTEIN-BINDING OF TROGLITAZONE STEREOISOMERS BY FLUOROMETRIC TITRATION

Determination of the protein binding of troglitazone is difficult beca use of its high adsorption to filters and membranes and the instabilit y of the stereoisomers. We attempted to assess the protein binding of four stereoisomers of troglitazone in the plasma and albumin from seve ral species by the method using fluorescent probes. The inhibition con stants (K-i) for the stereoisomers of troglitazone were obtained from the decreases in fluorescence intensity of dansylsarcosine caused by c ompetitive inhibition. Each stereoisomer of troglitazone displaced dan sylsarcosine, a typical specific fluorescent probe for the diazepam bi nding site on human serum albumin (HSA). The highest binding affinity for dansylsarcosine was observed with HSA (dissociation constant, K-d, K-1=0.5 mu M), while it was lowest in the mouse (K-d,K-1=18 mu M) The K-i values for KK and ddY mouse plasma and mouse and rat albumin were in the range 2-15 mu M, and there were no large variations among stere oisomers, the maximum differences being twofold, For human plasma and albumin, the displacement could not be accounted for by a simple compe titive inhibition. Comparison between unbound fraction (f(u)) values c alculated from thus obtained K-i values and those Df a mixture of the four stereoisomers determined by high-performance frontal analysis sho wed that the f(u) values obtained by fluorometric titration were highe r, while the relative differences among the stereoisomers in terms of animal species and strain were comparable for the two methods, Small d ifferences in protein binding among stereoisomers of troglitazone may not be the major reason for their stereoselective pharmacokinetics. (C ) 1997 by John Wiley & Sons, Ltd.