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 dissoci ation constants with which pharmaceutical candidates and drugs bind to seru m albumin and to alpha(1)-acid glycoprotein with the goal of deducing the e xtent of binding. Methods. The quenching of the intrinsic tryptophan fluorescence of serum al bumin and alpha(1)-acid glycoprotein was monitored by spectrofluorimetry an d the data were used to calculate the apparent dissociation constant. Sodiu m 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, iophenoxi c acid, theophylline, chloramphenicol, acetaminophen, lithium chloride and ampicillin were also investigated. Chlorpromazine hydrochloride and imipram ine hydrochloride were used as probes for alpha(1)-acid glycoprotein. The a ssays were also extended to the multiwell format. The quenching curves were fitted to the quadratic binding equation to determine the dissociation con stants. Results. Intrinsic fluorescence measurements are an excellent predictor of the drug binding to human serum albumin and to alpha(1)-acid glycoprotein. These measurements detect binding to the warfarin and benzodiazepine bindin g sites of human serum albumin. The dissociation constants estimated using the method compare favorably to the dissociation constants previously repor ted by Epps ct nl. using extrinsic fluorescence methodology, and the result s correlate well with equilibrium dialysis using drug displacement endpoint s. Conclusions. These measurements can be carried out with small samples and d o not require separation of the bound and unbound species. Additionally, th e proposed methods eliminate membrane separations, are not compound specifi c and do not require analytical chromatography of mass spectrometry for qua ntitation. Spectrofluorimetry may prove to be a useful method for rapidly d etermining the protein binding of combinatorial libraries.