Accumulation of anthracenyl-amino acid topoisomerase I and II inhibitors in drug-sensitive and drug-resistant human ovarian cancer cell lines determined by high-performance liquid chromatography

Abstract

Anthracenyl amino acid/dipeptide conjugates (AADC) represent novel structures rationally designed for their DNA-binding properties. A high-performance liquid chromatography method is described for simultaneous determination of five compounds that exhibit novel mechanisms of action as topoisomerase I and II inhibitors. The method uses an Apex ODS-2 column and a mobile phase of 0.25 M ammonium acetate/trifluoroacetic acid (pH 3) in methanol with gradient elution. Selective detection is achieved by monitoring at 545 nm, with limits of detection ranging between 2 and 4 ng on the column. AADC are recovered from cell sonicates by solid-phase extraction using C2 cartridges, with extraction efficiencies ranging from 84% to 95%. Drug uptake studies were performed with three active compounds in the human ovarian cancer cell line A2780 and its multi-drug-resistant counterpart 2780AD. Marked differences were observed in the pattern of cellular accumulation produced by each compound. NU/ICRF 505 (tyrosine derivative) was taken up most avidly, reaching plateau levels of 4000 pmol/10(6) cells after 2 h, with no difference being apparent between A2780 and 2780AD. NU/ICRF 510 (arginine derivative) accumulated slowly in A2780, failing to achieve an equilibrium after 4 h, and appeared to be completely excluded from 2780AD. NU/ICRF 500 (serine derivative) was most rapidly taken up by A2780, producing a plateau of 800 pmol/10(6) cells after only 30 min with approximately 3-fold less accumulation in 2780AD. These results are correlated to the chemosensitivity of the two cell lines to the three compounds.

The Design of Organometallic Ruthenium Arene Anticancer Agents

“…[17] This cell line displays the classic Multi Drug Resistance (MDR) phenotype via over-expression of the 170 kD plasma membrane glycoprotein P-gp and reduced cellular drug accumulation. [43,44] P-gp has a substrate specificity for naturally-occurring hydrophobic molecules, particularly those carrying a positive charge and so the cationic hydrophobic Ru II arene complexes exhibit both these features. Thus it is likely that the cross resistance to Ru II arene complexes is due, at least partly, to their recognition and active efflux by P-gp.…”

Topoisomerase I Poisons and Suppressors as Anticancer Drugs

“…For example, the anthraquinone drug NU/ICRF 505 (Fig. 9) is a potent topoisomerase I poison whereas closely related analogs with, for example, a phenylalanine in place of the tyrosine residue have no effect on topoisomerase I actitivity [104][105][106][107]. Binding of NU/ICRF 505 to DNA involves intercalation of the anthraquinone chromophore coupled with minor groove anchorage of the appended amino acyl moiety [108].…”

Determination of the glucuronide metabolites of the topoisomerase I inhibitors 7-Ethyl 10-hydroxycamptothecin (SN-38) and NU/ICRF 505 by high-performance liquid chromatography

“…Chromatographic conditions to resolve these two species have required the addition of ion-pairing agents and/or high ionic strength buffers to the mobile phase, making these methods incompatible with mass spectrometry [18,19,22]. In a previous report a mobile phase consisting of 0.25 M-ammonium acetate adjusted to pH 3 with 25% trifluoroacetic acid was necessary to resolve NU/ICRF 505 from its free amino acid hydrolysis product referred to as NU!…”

“…NU/ICRF 505 was synthesised in-house through the reaction of cz-tyrosine ester with (2H, 3H)-9,10-dihydroxyanthracene-1, 4-dione as part of a programme to rationally design novel topoisomerase I inhibitors, and was chemically characterised as previously reported [18]. NU/ICRF 505/M, the hydrolysis product of the C-terminal ethyl ester linkage producing the free amino acid, was synthesised, purified and chemically characterised as described previously [15].…”