Determination of protein binding of troglitazone stereoisomers by fluorometric titration.
Determination of the protein binding of troglitazone is difficult because of its high adsorption to filters and membranes and the instability of the stereoisomers. We attempted to assess the protein binding of four stereoisomers of troglitazone in the plasma and albumin from several species by the method using fluorescent probes. The inhibition constants (Ki) for the stereoisomers of troglitazone were obtained from the decreases in fluorescence intensity of dansylsarcosine caused by competitive inhibition. Each stereoisomer of troglitazone displaced dansylsarcosine, a typical specific fluorescent probe for the diazepam binding site on human serum albumin (HSA). The highest binding affinity for dansylsarcosine was observed with HSA (dissociation constant, Kd,1 = 0.5 microM), while it was lowest in the mouse (Kd,1 = 18 microM). The Ki values for KK and ddY mouse plasma and mouse and rat albumin were in the range 2-15 microM, and there were no large variations among stereoisomers, the maximum differences being twofold. For human plasma and albumin, the displacement could not be accounted for by a simple competitive inhibition. Comparison between unbound fraction (fu) values calculated from thus obtained Ki values and those of a mixture of the four stereoisomers determined by high-performance frontal analysis showed that the fu values obtained by fluorometric titration were higher, while the relative differences among the stereoisomers in terms of animal species and strain were comparable for the two methods. Small differences in protein binding among stereoisomers of troglitazone may not be the major reason for their stereoselective pharmacokinetics.
Izumi
T
T
Analytical and Metabolic Research Laboratories, Sankyo Co. Ltd., Tokyo, Japan.
Hoshiyama
K
K
Enomoto
S
S
Sasahara
K
K
Sugiyama
Y
Y
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002839:Chromans / Q000097:blood* / Q000493:pharmacokinetics; D003619:Dansyl Compounds / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007004:Hypoglycemic Agents / Q000097:blood* / Q000493:pharmacokinetics; D008297:Male; D051379:Mice; D011485:Protein Binding; D051381:Rats; D012521:Sarcosine / Q000031:analogs & derivatives / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism*; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013844:Thiazoles / Q000097:blood* / Q000493:pharmacokinetics; D045162:Thiazolidinediones; D017186:Titrimetry; D000077288:Troglitazone
D002839:Chromans; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D007004:Hypoglycemic Agents; D012709:Serum Albumin; D013844:Thiazoles; D045162:Thiazolidinediones; C037087:dansylsarcosine; D000077288:Troglitazone; D012521:Sarcosine