A rapid spectrofluorimetric technique for determining drug-serum protein binding suitable for high-throughput screening

Purpose. To develop and validate a rapid method for determining the dissociation constants with which pharmaceutical candidates and drugs bind to serum albumin and to α₁-acid glycoprotein with the goal of deducing the extent of binding. Methods. The quenching of the intrinsic tryptophan fluorescence of serum albumin and α₁-acid glycoprotein was monitored by spectrofluorimetry and the data were used to calculate the apparent dissociation constant. Sodium warfarin was used to probe the warfarin-binding site of serum albumin and diazepam was used to probe the benzodiazepine binding site. Additionally, the binding of sodium salicylate, phenylbutazone, sulfinpyrazone, iophenoxic acid, theophylline, chloramphenicol, acetaminophen, lithium chloride and ampicillin were also investigated. Chlorpromazine hydrochloride and imipramine hydrochloride were used as probes for α₁-acid glycoprotein. The assays were also extended to the multiwell format. The quenching curves were fitted to the quadratic binding equation to determine the dissociation constants. Results. Intrinsic fluorescence measurements are an excellent predictor of the drug binding to human serum albumin and to α₁-acid glycoprotein. These measurements detect binding to the warfarin and benzodiazepine binding sites of human serum albumin. The dissociation constants estimated using the method compare favorably to the dissociation constants previously reported by Epps et al. using extrinsic fluorescence methodology, and the results correlate well with equilibrium dialysis using drug displacement endpoints. Conclusions. These measurements can be carried out with small samples and do not require separation of the bound and unbound species. Additionally, the proposed methods eliminate membrane separations, are not compound specific and do not require analytical chromatography or mass spectrometry for quantitation. Spectrofluorimetry may prove to be a useful method for rapidly determining the protein binding of combinatorial libraries.