HPLC methods for the determination of bound and free doxorubicin, and of bound and free galactosamine, in methacrylamide polymer-drug conjugates.

Quantitative acid hydrolysis followed by HPLC separation has been established as an analytical procedure for the determination of polymer-bound doxorubicin (an anti-cancer drug) and D-galactosamine (a liver-targetting moiety) in the polymer-drug conjugates FCE 28068 and FCE 28069. Optimal conditions of hydrolysis were determined in both cases: 1 N HCl, 50 degrees C, 1.5 h for doxorubicin, and 6 N HCl, 60 degrees C, 5 h for galactosamine. Appropriate HPLC quantitation of galactosamine required pre-treatment with sodium borohydride and pre-column derivatization with o-phthalalaldehyde and beta-mercaptoethanol. Independent determination of free doxorubicin and glactosamine in untreated polymer samples was also achieved with the same HPLC method up to detection limits of 0.01% and 0.02% respectively. The methods were validated for linearity, precision and repeatability. Validation for accuracy before and after acid hydrolysis was achieved by testing hydrolysis on model compounds and by assessing recovery in polymer solutions spiked with free doxorubicin or galactosamine.

Determination of phenolic ionization constants of anthracyclines with modified substitution pattern of anthraquinone chromophore.

The phenolic ionization constants in aqueous solution of doxorubicin, daunorubicin and nine daunorubicin analogues with different substitution patterns at the anthraquinone moiety have been determined spectrophotometrically upon taking into account aggregation effects with extrapolation to infinite dilution. In contrast with an early literature report [Sturgeon, R.J. & Schulman, S.G. J. Pharm. Sci. 66, 958-961; 1977] the perturbation from amino group ionization on daunosamine sugar was found to be negligible in our spectrophotometric titrations. Accordingly, only phenol hydroxyl ionization constants could be determined in these experiments, but a crude estimate of daunosamine pKa was obtained in a fluorometric titration. From a comparison of suitable analogues it is concluded that the phenolic function at C11 in doxorubicin and daunorubicin is the most acidic one. The higher pKa of the C6-OH group is ascribed in part to electronic effects leading to higher strength of the corresponding hydrogen bond with quinone oxygen, and in part to a steric effect from the bulky sugar group at C7. Ionization constants of nitro or amino substituted derivatives follow the expected trend. In the cases of carminomycin and 6-deoxycarminomycin, which both have another phenolic group at C4, two phenolic ionization processes can be detected in the experimentally accessible pH range (5-12): these are ascribed to C4-OH and C11-OH. An application of the compiled parameters to studies of chemical reactivity and biological activity of anthracyclines is foreseen.

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity.

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.