THE STRUCTURAL BASIS OF CAMPTOTHECIN INTERACTIONS WITH HUMAN SERUM-ALBUMIN - IMPACT ON DRUG STABILITY

The intense intrinsic fluorescence emissions from several clinically r elevant camptothecin drugs have been exploited in order to study the s tructural basis of drug binding to human serum albumin. Both HPLC and time-resolved fluorescence spectroscopic methodologies were employed t o characterize the associations of camptothecins with HSA in phosphate -buffered saline (pH 7.4) at 37 degrees C. The alpha-hydroxy delta-lac tone ring moiety of camptothecin (C), 10-hydroxycamptothecin (HC), 10, 11-(methylenedioxy) camptothecin (MC) and 9-chloro-10,11-(methylenedio xy) camptothecin (CMC) was in each case observed to hydrolyze more rap idly and completely in the presence of HSA than in the protein's absen ce. Binding isotherms constructed by the method of fluorescence lifeti me titration showed that HSA bound preferentially the carboxylate form s of C, HC, MC, and CMC over their lactone forms, thereby providing an explanation for the shift to the right in the lactone-carboxylate equ ilibrium observed for each compound upon HSA addition. In marked contr ast, three analogues (SN-38, CPT-11, and topotecan) all displayed enha nced stabilities in the presence of HSA. While the lifetimes of CPT-11 , topotecan, and the carboxylate forms of both drugs were insensitive to the addition of HSA, the lifetimes of both SN-38 and its carboxylat e form did titrate upon HSA addition. Analysis of binding isotherms co nstructed for the albumin interactions of SN-38 and its carboxylate fo rm demonstrated a higher overall association constant for the lactone form [640 (M amino acid (aa) residues)(-1)] relative to the carboxylat e form [150 (M aa)(-1)]. Our studies indicate that specific modificati ons at the 7- and 9-positions of the quinoline nucleus, such as those contained in CPT-11, topotecan, and SN-38, enhance drug stability in t he presence of HSA. In the case of SN-38, the enhanced stability was s hown to be due to preferential associations between the drug's lactone form and the blood protein.