Platinum group metallodrug‐protein binding studies by capillary electrophoresis – inductively coupled plasma‐mass spectrometry: A further insight into the reactivity of a novel antitumor ruthenium(III) complex toward human serum proteins

Abstract

Biochemical speciation analysis has become a hot area of CE research due largely to growing emergence of inductively coupled plasma (ICP)-MS as a proper detection technique. A benefit of CE-ICP-MS coupling in species-selective analysis of anticancer metal-based drugs is the possibility of distinguishing the signals of the intact drug and its metabolites and hence of quantifying them independently. This advantage (over CE with UV-vis detection) was exploited here in order to gain better knowledge about the rate and degree of the transformation of indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), a promising tumor-inhibiting agent that successfully finished phase I clinical studies, upon its binding toward individual serum transport proteins. At increasing the KP1019/protein molar ratio, the reaction rate expressed by an evolving peak of the protein adduct became faster, with the equilibrium state being reached after about 40 and 60 min of incubation at 37 degrees C for transferrin and albumin, respectively. The binding reaction was shown to obey the first-order character that enabled for reliable calculation of the corresponding rate constants as (28.7 +/- 1.5) x 10(-4) and (10.6 +/- 0.7) x 10(-4)/s, respectively. When incubated with a ten-fold excess of KP1019, albumin and transferrin bound, respectively, up to 8 and 10 equiv. of ruthenium (Ru). Relative affinity of KP1019 toward different proteins under simulated physiological conditions was also characterized in terms of the overall binding constants (5600 and 10 600/M, respectively). To emphasize the difference in the protein-binding behavior, a competitive interaction of KP1019 was followed by CE-ICP-MS at the actual molar ratio of proteins in blood, i.e. a ten-fold excess of albumin over transferrin. The fact that KP1019 binds to albumin stronger than to transferrin was manifested by finding almost all ruthenium (98-99%) in the albumin fraction.

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

“…KP1019 is known to bind to a large degree to HSA, which is suggested to act as a ruthenium reservoir [15,21,22]. HSA is the most abundant of the blood serum proteins occurring to the extent of 0.63 mM.…”

“…A binding ratio of four KP1019 complex anions on a single HSA molecule was determined by CD and confirmed by inductively coupled plasma atomic emission spectroscopic (ICP-AES) method [23]. More recently, the KP1019−HSA association constants were determined by means of capillary electrophoresis (CE) as 9.9 × 10 3 M -1 , and by CE coupled to ICP-mass spectrometry (MS) as 1.06 × 10 4 M -1 [13,21]. It is noteworthy that separation methods such as CE may shift the chemical equilibrium of a reaction mixture and therefore spectroscopic methods provide a better means to investigate binding constants.…”

Capillary electrophoresis hyphenated to inductively coupled plasma‐mass spectrometry: A novel approach for the analysis of anticancer metallodrugs in human serum and plasma

“…It has been applied to investigate the stability of novel compounds but also the interactions of drugs with biomolecules, such as human serum proteins [8][9][10][11][12][13][14][15] and nucleotides [16][17][18][19][20][21], elucidating possible metabolic and reaction pathways of the drug candidates [6,7,16], notably always working under simulated physiological conditions with regard to pH, temperature, aqueous solutions, and/or buffer composition.…”

The application of inductively coupled plasma mass spectrometry in clinical pharmacological oncology research

“…The inconsistent results point out that, when developing a new assay, it is advisable to test several ISs for each matrix. For the analysis of Ru (IP: 7.4), yttrium ( 98 Y, IP; 6.4) and germanium ( 72 Ge, IP; 7.9) have been used as IS (Polec-Pawlak et al, 2006;Brouwers et al, 2007c). They both corrected well for matrix effects.…”

“…The Pt content of the bands was measured by ICP-MS. Timerbaev et al (2004) coupled capillary electrophoresis to ICP-MS using an interface consisting of a microconcentric nebuliser to study the interaction of cisplatin with albumine. A similar method was applied to study the interaction of KP1019 with albumine and transferrin (Polec-Pawlak et al, 2006).…”