Two dimensional separation schemes for investigation of the interaction of an anticancer ruthenium(iii) compound with plasma proteins

Abstract

On-line 2-dimensional size exclusion/anion exchange chromatography was coupled to inductively coupled mass spectrometry with dynamic reaction cell technology (SEC-IC-ICP-MS) in order to characterize the interaction of the ruthenium-based anticancer drug KP1019 with human plasma proteins in vitro and, for the first time, in vivo. In SEC-ICP-MS studies the drug was found to bind exclusively to the protein fraction of 60–80 kDa in human plasma samples (clinical study/phase 1). The respective size fraction was collected and subsequently analyzed by reversed phase chromatography coupled to electrospray mass spectrometry (LC-ESI-MS) confirming the presence of the two well known transporter proteins, i.e. human serum albumin (HSA) and transferrin (Tf). Hence, for in vivo investigation of KP1019 interaction with HSA and Tf a fully automated SEC-IC-ICP-MS approach was applied. The stoichiometry of the KP1019 protein binding was determined through the molar Ru/S ratio. Human apo-Tf standards incubated with different stoichiometric equivalents of KP1019 were used for species-specific and species-unspecific calibration of the molar Ru/S ratio. Competitive in vitro incubation of KP1019 to both HSA and Tf for ca. 10 h showed that <20% of the overall KP1019 was found to bind to Tf in an equimolar mixture of Tf and HSA. This fraction dropped to 2% in the incubated mixture containing a 10-fold excess of HSA compared to Tf. This implied that less than 2% of KP1019 were expected to bind to Tf in human plasma samples, as they usually contain an even higher excess of HSA. Indeed, the concentration of the KP1019/Tf-adduct was below the limit of quantification in the plasma sample taken from a cancer patient treated with KP1019. Its fraction of the overall KP1019-content was estimated to be <1%. The KP1019/HSA stoichiometry in the plasma samples could be correlated with the individual infusions administered to the cancer patient within 26 days, the largest value being 1.4 (in the related sample, the KP1019/Tf stoichiometry was estimated to be in the range of 0.3–0.5). The KP1019/Tf ratio is in the expected range for a sufficient uptake via the transferrin pathway.