DIFFERENTIAL INTERACTIONS OF CAMPTOTHECIN LACTONE AND CARBOXYLATE FORMS WITH HUMAN BLOOD COMPONENTS

The intrinsic fluorescent emissions from the lactone and carboxylate f orms of camptothecin have been exploited in order to elucidate their m arkedly different interactions with the various components of human bl ood. In phosphate-buffered saline (PBS) at pH 7.4, human serum albumin (HSA) preferentially binds the carboxylate form with a 150-fold highe r affinity than the lactone form; these interactions result in camptot hecin opening more rapidly and completely in the presence of HSA than in the protein's absence [Burke, T. G., and Mi, Z. (1993) Anal. Bioche m. 212, 285-287]. In human plasma, at pH 7.4 and 37 degrees C, we have observed camptothecin lactone to open rapidly and fully to the carbox ylate form (t(1/2) = 11 min; % lactone at equilibrium, 0.2%). Substitu tion of a 10-hydroxy moiety into the camptothecin fluorophore makes th e agent's emission spectrum highly sensitive to microenvironment polar ity; we have observed pronounced blue shifting (from 530 to 430 nm) in the emission spectra of the hydroxy-substituted carboxylate both upon HSA association as well as upon drug dissolution in organic solvents of low dielectric strength. Hence, it appears that camptothecin carbox ylate's fluorophore locates in a hydrophobic binding pocket in native HSA. Ionic interactions also appear to strongly affect binding between camptothecin carboxylate and the HSA binding pocket, since a 6-fold i ncrease in solution salt concentration diminished camptothecin carboxy late binding by 10-fold. Our findings that HSA denaturation abolishes high-affinity binding indicate that interactions of the carboxylate dr ug form are specific for the native HSA conformation. Interestingly, h igh-affinity binding of the carboxylate appeared not to occur in the p resence of other blood proteins, such as gamma-globulin, alpha(1)-acid glycoprotein, fibrinogen, and the oxy and deoxy forms of hemoglobin. In whole blood versus plasma, camptothecin was found to display enhanc ed stability (t(1/2) value of 22 min and a lactone concentration at eq uilibrium value of 5.3%). The enhanced stability of camptothecin in hu man blood was found to be due to drug associations with the lipid bila yers of red blood cells. Camptothecin lactone partitions into the lipi d bilayers of erythrocytes, with the drug locating in a hydrophobic en vironment protected from hydrolysis.