Protolytic equilibria of doxorubicin as determined by isoelectric focusing and ‘electrophoretic titration curves’

Doxorubicin (AdriamycinR) and the related compound daunorubicin (Daunomycin) are antibiotics of the anthracycline group [l] with cytotoxic and antimitotic activity [2]. Their clinical uses in the treatment of acute leukemia and solid tumors have been reviewed [3-51. Their biological activity is linked to the formation of an intercalative complex with DNA [6-S] with concomitant i~libition of both RNA and DNA synthesis (9,101. Doxorubicin is also known to interact with phospholipids [ 11 ,I 2) and with sulphated mucopolysaccharides [ 131. On the other hand, Daunorubicin has been shown to bind to non-histone protein from rat liver [ 141 and, due to the presence of planar aromatic rings in its quinone moiety, to interact strongly with itself [ 151, forming mostly dimers and tetramers, as already shown for many similar compounds like actinomycin, proflavin, purines or acridine orange. This self-association, in turn, should affect the acid dissociation constants of the two groups ionizable at or near physioiogic~ pH, i.e., the 3’-NHz group in the sugar moiety and the 11 -OH group in the quinone ring. This could explain why literature data on the amino group pK values varyfrom7.2-8.99 [1,16,17] andfrompK9.54-10.1 for the phenolic -OH [ 16,181. These pK’ values have been determined by potentiometric (amino group) as well as by spectrophotometric and fluorimetric (‘phenolic -OH) titrations and, more recently, on the basis of partition coefficients in chloroform-l pentanol [16]. In this report, we have attempted to measure protolytic equilibria of doxorubicin indirectly, by equilibrium isoelectric focusing, by p1 and Apl value determinations, and directly, by using ‘electrophoretic titration curves’. This last technique, described [ 19,201, has been successfully applied to the study of macromolecule-ligands [21] and macromoleculemacromolecule interactions [22].